Method for testing and screening p38 map kinase modifiers

ABSTRACT

This invention provides methods for treating diseases associated with elevated p38 mitogen-activated protein kinase activity. Moreover, the invention provides methods for testing a candidate compound for a p38 mitogen-activated protein kinase modifying activity by calculating the level of relocalization of an SMN complex component from the cytoplasm to the nucleus of a cell. Additionally, the invention provides a kit and a system for calculating the same.

FIELD OF INVENTION

This invention provides: Compounds that inhibit p38 mitogen-activatedprotein kinase, methods, kits, and systems for testing a candidatecompound for p38 mitogen-activated protein kinase (MAPK) modifyingactivity.

BACKGROUND OF THE INVENTION

Translation is the RNA directed synthesis of polypeptides. This processrequires all three classes of RNA. Although the chemistry of peptidebond formation is relatively simple, the processes leading to theability to form a peptide bond are exceedingly complex. The template forcorrect addition of individual amino acids is the mRNA, yet both tRNAsand rRNAs are involved in the process. The tRNAs carry activated aminoacids into the ribosome which is composed of rRNA and ribosomalproteins. The ribosome is associated with the mRNA ensuring correctaccess of activated tRNAs and containing the necessary enzymaticactivities to catalyze peptide bond formation.

Regulation of this information pathway can be achieved at a number oflevels, including the modulation of translation factor levels oractivity, ribosome biogenesis, and small molecule/RNA interactions.Small molecule ligands that inhibit the process of translation provideexquisite insight into ribosome function and translation factor activityin both prokaryotes and eukaryotes.

Inhibitors targeting a specific step of protein synthesis enabledissection of the translation pathway by allowing the characterizationof events leading to the assembly of active polysomes, trappingintermediates of the initiation and elongation cycle, and providinginsight into the molecular functions of protein factors.

Deregulation of protein synthesis is a major contributor in cancerinitiation and metastatic progression. Overexpression of some initiationfactors can lead to malignant transformation, whereas down-regulation ofthese same factors can suppress the transformed phenotype. In cancerscomponents of the translation apparatus are overexpressed or mutated.For example, the tumor suppressor gene product pRB directly impacts onthe translation process by affecting the levels of ribosomes.Furthermore, key components of anti-apoptotic pathways aretranslationally regulated.

Thus, protein synthesis represents a valid target for chemotherapeuticintervention. Few inhibitors of protein synthesis have been tested inclinical trials, with some of these demonstrating encouragingtherapeutic effects. Presumably, a therapeutic index is achieved due tothe higher requirement of transformed cells for protein synthesis, aswell as translation regulation of some of the proteins involved incancer progression. Unfortunately, dose-limiting secondary effects havehampered further development of many of these compounds.

Mitogen-activated protein kinases (MAPKs) are a family ofserine/threonine protein kinases that mediate fundamental biologicalprocesses and cellular responses to external stress signals. Increasedactivity of MAPK, in particular p38 MAPK, and their involvement in theregulation of the synthesis of inflammation mediators at the level oftranscription and translation, make them potential targets foranti-inflammatory therapeutics. Inhibitors targeting p38 MAPK and JNKpathways exhibit anti-inflammatory activity.

p38 is a major signal transducer responding to cellular stress stimulisuch as cytokines. p38 was independently identified by multiple groupswho were isolating kinases involved in cellular responses to cellularstresses such as heat shock, osmotic stress, sodium arsenite andlipopolysaccharide (LPS). One of these research groups isolated andcloned human p38 by identifying the molecular target of a small-moleculeinhibitor of interleukin 1 (IL-1) and tumor necrosis factor α (TNF-α)production in response to LPS. This suggested not only the amenabilityof p38 as a drug target, but also the crucial role this pathway plays inmediating responses to cellular stress stimuli. Because p38 MAPKregulates the production of TNF-α and IL-1, p38 inhibitors are expectedto inhibit not only the production of pro-inflammatory cytokines, butalso their actions, thereby interrupting the vicious cycle that oftenoccurs in inflammatory and immunoresponsive diseases. Thus, p38 plays akey role in mediating cell survival, growth, differentiation,inflammation and innate immunity. p38 inhibitors may treat a wide rangeof indications, including cancer and arthritis.

SUMMARY OF THE INVENTION

This invention provides, in one embodiment, method for decreasing p38mitogen-activated protein kinase (MAPK) enzymatic activity in a cell,comprising the step of contacting a cell with a compound having astructure according to Formula (I):

Wherein R₁ and R₂ are independently substituted or unsubstituted,carbocyclic or heterocyclic aromatic ring; andX₁ and X₂ are independently O or S, thereby decreasing p38mitogen-activated protein kinase (MAPK) enzymatic activity in a cell.

In another embodiment, the present invention provides a method oftreating or inhibiting a disease in a subject, comprising administeringto said subject a compound having a structure according to Formula (I):

Wherein R₁ and R₂ are independently substituted or unsubstituted,carbocyclic or heterocyclic aromatic ring; and X₁ and X₂ areindependently O or S, wherein said disease is associated with elevatedp38 mitogen-activated protein kinase (MAPK) enzymatic activity in a cellin said subject, thereby treating or inhibiting a disease in a subject.

In another embodiment, the present invention provides a method fortesting a p38 mitogen-activated protein kinase (MAPK) modifier,comprising the steps of (a) contacting a cell with a candidate compoundand an additional compound, wherein additional compound is capable ofinhibiting protein synthesis in a cell and increasing an enzymaticactivity of a p38 MAPK in a cell; (b) and calculating the level ofrelocalization of an SMN complex component of a cell from the cytoplasmto the nucleus of a cell. In another embodiment, a method for testingfor a p38 MAPK modifier comprises a method for screening for a p38 MAPKmodifier. In another embodiment, a method for testing for a p38 MAPKmodifier comprises a method for testing for a p38 MAPK modifier.

In another embodiment, the present invention provides a method fortesting an ability of a candidate compound to inhibit a p38 MAPKactivity, comprising the steps of (a) contacting a cell with a proteinsynthesis inhibitor, a candidate compound, and an activator of p38 MAPKactivity; and (b) calculating the level of relocalization of an SMNcomplex component of a cell from the cytoplasm to the nucleus of a cell.

In another embodiment, the present invention provides a method forscreening or testing for a p38 mitogen-activated protein kinase (MAPK)activator, comprising the steps of contacting a cell with a candidatecompound and an additional compound, wherein the additional compound iscapable of inhibiting protein synthesis in a cell and calculating thelevel of relocalization of an SMN complex component of a cell from thecytoplasm to the nucleus of a cell.

In another embodiment, the present invention provides a kit forscreening for a modifier of p38 mitogen-activated protein kinase (MAPK)activity in a cell, comprising a means of measuring a relocation of anSMN complex component in a cell. In another embodiment, the presentinvention provides a kit for testing for a modifier of p38mitogen-activated protein kinase (MAPK) activity in a cell, comprising ameans of measuring a relocation of an SMN complex component in a cell.

In another embodiment, the present invention provides a system forscreening for a p38 MAPK modifier in a cell, comprising a kit forscreening a modifier of p38 mitogen-activated protein kinase (MAPK)activity in a cell, comprising a means of measuring a relocation of anSMN complex component in a cell. In another embodiment, the presentinvention provides a system for testing for a p38 MAPK modifier in acell, comprising a kit for screening a modifier of p38 mitogen-activatedprotein kinase (MAPK) activity in a cell, comprising a means ofmeasuring a relocation of an SMN complex component in a cell.

BRIEF DESCRIPTION OF THE DRAWINGS

The subject matter regarded as the invention is particularly pointed outand distinctly claimed in the concluding portion of the specification.The invention, however, both as to organization and method of operation,together with objects, features, and advantages thereof, may best beunderstood by reference to the following detailed description when readwith the accompanying drawings in which:

FIG. 1A shows an immunostaining micrograph [×40] of SMN followingtreatment with RNAi of a non-targeting siRNA (A), following RNAi of SMN(B), RNAi of Gemin2 (C), RNAi of Gemin3 (D), RNAi of Gemin4 (E) and RNAiof Gemin5 (F). FIG. 1B depicts a bar graph which represents quantitationof the average anti-SMN (2B1) antibody signal intensities per Gem (blackbars) and the average numbers of Gems per cell (white bars) of theimages shown in FIG. 1A.

FIG. 2A and FIG. 2B shows an immunostaining micrograph [×20] identifyingalterations in SMN sub-cellular localization. FIG. 2A showsimmunostaining of SMN in DMSO-treated cells. FIG. 2B showsimmunostaining of SMN in cells treated for 4 hours with a compound fromthe library that was identified as having a significant change in SMNsub-cellular distribution. FIG. 2C shows a bar graph which representsquantitation of the average changes in cytoplasmic fluorescenceintensity in control (DMSO treated cells) (black bars) and in cellstreated for 4 hours with a compound having a significant change in SMNsub-cellular distribution (white bars).

FIG. 3A shows an immunostaining micrograph of SMN in control HeLa PVcells and following treatment with 10 μM cycloheximide (CHX) for theindicated times. Control cells were treated with DMSO at the same finalconcentration as that used to dissolve CHX. FIG. 3B shows a microscopicimmunostaining micrograph of SMN localization in HeLa cells treated with5-20 μM cycloheximide and immunostained after 4 hours of treatment. FIG.3C shows a microscopic immunostaining micrograph of FXR1, PABP, hnRNP A1and Sm proteins (snRNPs) in control HeLa cells (upper panel) and after 6hrs of 10 μM CHX treatment (lower panel).

FIG. 4 shows an immunostaining micrograph of sub-cellular localizationof an SMN complex component in control HeLa cells and in CHX treatedcells Immunostaining of Gemins 2, 3, 5, 6 and 7 in control DMSO treatedcells is shown in the upper panel Immunostaining of Gemins 2, 3, 5, 6and 7 in cells treated for 6 hours with 10 μM CHX is shown in the lowerpanel.

FIG. 5 shows an immunostaining micrograph of sub-cellular localizationof an SMN complex component in control HeLa cells and in HDAC inhibitorstreated cells. FIG. 5A shows immunostaining of SMN in DMSO treated cells(control) and in cells treated with 2 μM TSA, 7.5 mM VPA, 8 μMscriptaid, 10 μM HDAC inhibitor I and 5 μM apicidin for 24 hours. FIG.5B shows immunostaining of SMN in HeLa cells treated with 2-10 mM VPA.FIG. 5C shows immunostaining of Gemins 2, 3, 5, and 6 in control cells(upper panel) and in cells treated for 24 hrs with 10 mM VPA (lowerpanel).

FIG. 6 shows a microscopic immunostaining micrograph of sub-cellularlocalization of an SMN complex component in control HeLa cells and intreated HeLa cells (after 6 hours of treatment). Upper panel: Controlshows immunostaining of SMN in cells treated with DMSO. CHX showsimmunostaining of SMN in cells treated with 10 μM CHX. Anisomycin showsimmunostaining of SMN in cells treated with 1 μg/ml Anisomycin. TNFαshows immunostaining of SMN in cells treated with 150 ng/ml TNFα. Lowerpanel: CHX+Anisomycin shows immunostaining of SMN in cells treated with10 μM CHX+10 μM Anisomycin. CHX+SB202190 shows immunostaining of SMN incells treated with 10 μM. Anisomycin+10 μM SB202190 Anisomycin+SB202190shows immunostaining of SMN in cells treated with 1 μg/ml Anisomycin+10μM SB202190. TNFα+CHX shows immunostaining of SMN in cells treated with10 μM CHX+150 ng/ml TNFα.

FIG. 7 schematically depicts the suggested mechanism controlling SMNcomplex relocalization from the cytoplasm to the nucleus. Underconditions which inhibit protein translation SMN complex relocalizesfrom the cytoplasm to the nucleus. SMN complex relocalizes from thecytoplasm to the nucleus also under conditions which induceendoplasmatic reticulum (ER) stress. Activated p38 MAPK inhibits CHXinduced SMN complex relocalization from the cytoplasm to the nucleus.SB202190 and SB203580 inhibit the activation and activity of p38 MAPK;thus, SMN complex relocalizes from the cytoplasm to the nucleus as aresult of protein synthesis inhibition by CHX.

FIG. 8 shows an immunostaining micrograph of SMN localization. FIG. 6ais immunostaining of SMN in control HeLa cells (left panel) and in cellstreated with an inactive analogue of cycloheximide,cycloheximide-N-ethylethanoate (CHX-N) at 10 μM. FIG. 6b isimmunostaining of SMN showing nuclear accumulation in cells treated withemetine 10 μM, puromycin 10 μM, thapsigargin 10 μM, and tunicamycin 10μg/ml, as depicted on the panels.

FIG. 9 shows an immunostaining micrograph of SMN localization in HeLa PVcells stably transfected with a non-targeting shRNA (upper panels) andin cells stably transfected with an shRNA targeting SMN (lower panels).Both cell lines were treated with DMSO as control at the same finalconcentration as that used to dissolve CHX and following treatment with10 μM cycloheximide (CHX) for 6 hours as indicated.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides, in one embodiment, a compound having astructure according to Formula (I):

Wherein R₁ and R₂ are independently substituted or unsubstituted,carbocyclic or heterocyclic aromatic ring; andX₁ and X₂ are independently O or S.

In another embodiment, provided herein a compound having a structureaccording to Formula (I):

Wherein R₁ and R₂ are independently substituted or unsubstituted,carbocyclic or heterocyclic aromatic ring; andX₁ is S and X₂ is O. In another embodiment, Xi is a heterocompoundcapable of maintaining the functionality of the p38 inhibitors describedherein, such as Selenium in one embodiment, or a Sleenium-containingcompound.

In another embodiment, the compound of formula I comprises R₁ and R₂comprising 5-8 membered aromatic ring. In another embodiment, thecompound of formula I comprises R₁ and R₂ comprising 5-6 memberedaromatic ring. In another embodiment, the compound of formula Icomprises R₁ and R₂ comprising 5 and 6 membered aromatic ring.

In another embodiment, the compound of formula I comprises aheterocyclic aromatic ring comprising N, S, O, or a combination thereof.In another embodiment, the compound of formula I comprises aheterocyclic aromatic ring comprising at least a single N. In anotherembodiment, the compound of formula I comprises a heterocyclic aromaticring comprising at least a single S. In another embodiment, the compoundof formula I comprises a heterocyclic aromatic ring comprising at leasta single O. or a combination thereof.

In another embodiment, the compound of formula I comprises a structureaccording to Formula (II):

In another embodiment, the compound of formula I comprises a structureaccording to Formula (III):

In another embodiment, the compound of formula I comprises a structureaccording to Formula (IV):

In another embodiment, the compound of formula I comprises a structureaccording to Formula (V):

In another embodiment, the compound of formula I is a p38mitogen-activated protein kinase (MAPK) modifier. In another embodiment,the compound of formula I is a MAPK inhibitor. In another embodiment,the compound of formula I is a p38 inhibitor.

In another embodiment, the compound of formula I is used in aformulation. In another embodiment, a formulation comprising thecompound of formula I is used to inhibit tumor growth. In anotherembodiment, a formulation comprising the compound of formula I is usedto inhibit metastasis. In another embodiment, a formulation comprisingthe compound of formula I is used to treat a subject afflicted withcancer as described hereinunder. In another embodiment, a formulationcomprising the compound of formula I is used to treat a subjectafflicted with breast cancer. In another embodiment, a formulationcomprising the compound of formula I is used to treat a subjectafflicted with Alzheimer's disease. In another embodiment, a formulationcomprising the compound of formula I is used to treat a subjectafflicted with early stages of Alzheimer's disease. In anotherembodiment, a formulation comprising the compound of formula I is usedto treat a subject afflicted with amyotrophic lateral sclerosis (ALS orLou Gehrig's disease). In another embodiment, a formulation comprisingthe compound of formula I is used to treat a subject afflicted withearly stages of ALS (Lou Gehrig's disease). In another embodiment,amyotrophic lateral sclerosis and Alzheimer's disease are associatedwith elevated p38 mitogen-activated protein kinase (MAPK) enzymaticactivity in diseased cells. In another embodiment, amyotrophic lateralsclerosis and Alzheimer's disease are characterized by elevated p38mitogen-activated protein kinase (MAPK) enzymatic activity in diseasedcells.

In another embodiment, a formulation comprising the compound of formulaI is used to treat a subject afflicted with an Encephalomyocarditisvirus. In another embodiment, a formulation comprising the compound offormula I is used to treat a subject afflicted with Hepatitis C virus.In another embodiment, a formulation comprising the compound of formulaI is used to treat a subject infected with HIV. In another embodiment, aformulation comprising the compound of formula I is used to treat asubject afflicted with AIDS. In another embodiment, cells infected oraffected by Hepatitis C virus comprise elevated p38 mitogen-activatedprotein kinase (MAPK) enzymatic activity. In another embodiment, cellsinfected or affected by Encephalomyocarditis virus comprise elevated p38mitogen-activated protein kinase (MAPK) enzymatic activity. In anotherembodiment, HIV and AIDS are diseases characterized by elevated p38mitogen-activated protein kinase (MAPK) enzymatic activity in diseasedcells.

In another embodiment, provided herein a method for decreasing p38mitogen-activated protein kinase (MAPK) enzymatic activity in a cell,comprising the step of contacting a cell with a compound having astructure according to Formula (I):

Wherein R₁ and R₂ are independently substituted or unsubstituted,carbocyclic or heterocyclic aromatic ring; andX₁ and X₂ are independently O or S, thereby decreasing p38mitogen-activated protein kinase (MAPK) enzymatic activity in a cell.

In another embodiment, the present invention provides a method oftreating or inhibiting a disease in a subject, comprising administeringto a subject a compound having a structure according to Formula (I):

Wherein R₁ and R₂ are independently substituted or unsubstituted,carbocyclic or heterocyclic aromatic ring; and X₁ and X₂ areindependently O or S, wherein said disease is associated with elevatedp38 mitogen-activated protein kinase (MAPK) enzymatic activity in a cellin said subject, thereby treating or inhibiting a disease in a subject.In another embodiment, the present invention provides a method oftreating or inhibiting a disease in a subject characterized by cellshaving elevated p38 mitogen-activated protein kinase (MAPK) enzymaticactivity in a cell. In another embodiment, the present inventionprovides a method of reducing the symptoms associated with a diseasecharacterized by cells having elevated p38 mitogen-activated proteinkinase (MAPK) enzymatic activity in a cell. In another embodiment, thepresent invention provides a method of reducing elevated p38mitogen-activated protein kinase (MAPK) enzymatic activity in a cellthereby treating a disease associated with elevated p38mitogen-activated protein kinase (MAPK) enzymatic activity in a cell. Inanother embodiment, the present invention provides a method of reducingelevated p38 mitogen-activated protein kinase (MAPK) enzymatic activityin a cell thereby treating a disease characterized by elevated p38mitogen-activated protein kinase (MAPK) enzymatic activity in a cell.

In another embodiment, provided herein a method for testing a p38mitogen-activated protein kinase (MAPK) modifier, comprising the stepsof (a) contacting a cell with a candidate compound and an additionalcompound, wherein additional compound is capable of inhibiting proteinsynthesis in a cell and increasing an enzymatic activity of a p38 MAPKin a cell; and (b) measuring the level of a SMN complex component in thecytoplasm of the cell, the nucleus of the cell, or a combinationthereof, whereby, if the candidate compound increases the amount of theSMN complex component in the nucleus, decreases or inhibits the amountof the SMN complex component in the cytoplasm, or a combination thereof,then the compound exhibits an ability to decrease or inhibit MAPKenzymatic activity. In another embodiment, a control cell is contactedwith the additional compound but not the candidate compound. In anotherembodiment, increase of the SMN complex component in the nucleus and/ordecrease or inhibit in the cytoplasm is calculated relative to thecontrol cell. Each possibility represents a separate embodiment of thepresent invention.

In another embodiment, the present invention provides a method fortesting an ability of a candidate compound to inhibit a p38 MAPKactivity pathway, comprising the steps of (a) contacting a cell with aprotein synthesis inhibitor, a candidate compound, and an activator ofp38 MAPK activity pathway; and (b) measuring the level of a SMN complexcomponent in the cytoplasm of the cell, the nucleus of the cell, or acombination thereof, whereby, if the candidate compound increases theamount of the SMN complex component in the nucleus, decreases orinhibits the amount of the SMN complex component in the cytoplasm, or acombination thereof, then the compound exhibits an ability to decreaseMAPK enzymatic activity. In another embodiment, a control cell iscontacted with the protein synthesis inhibitor and the activator of p38MAPK activity, but not the candidate compound. In another embodiment,increase of the SMN complex component in the nucleus and/or decrease inthe cytoplasm is calculated relative to the control cell. Eachpossibility represents a separate embodiment of the present invention.In another embodiment, the present invention provides a method fortesting an ability of a candidate compound to inhibit consequences of ap38 MAPK activity pathway. In another embodiment, the present inventionprovides a method for testing an ability of a candidate compound toinhibit p38 MAPK downstream signaling events known to one skilled in theart. In another embodiment, the present invention provides a method fortesting an ability of a candidate compound to inhibit p38 MAPK upstreamsignaling events known to one skilled in the art.

In another embodiment, the present invention provides a method forscreening for a p38 mitogen-activated protein kinase (MAPK) activator,comprising the steps of contacting a cell with a candidate compound andan additional compound, wherein the additional compound is capable ofinhibiting protein synthesis in a cell, and calculating the level ofrelocalization of an SMN complex component of a cell from the cytoplasmto the nucleus of a cell. In another embodiment, the present inventionprovides a method for testing a candidate compound for a p38mitogen-activated protein kinase (MAPK) activator, comprising the stepsof contacting a cell with a candidate compound and an additionalcompound, wherein the additional compound is capable of inhibitingprotein synthesis in a cell, and calculating the level of relocalizationof an SMN complex component of a cell from the cytoplasm to the nucleusof a cell.

In another embodiment, a control cell is contacted with the additionalcompound but not the candidate compound. In another embodiment, increaseof the SMN complex component in the nucleus and/or decrease in thecytoplasm is calculated relative to the control cell. Each possibilityrepresents a separate embodiment of the present invention.

In another embodiment, the present invention provides a kit forscreening for a modifier of p38 mitogen-activated protein kinase (MAPK)activity in a cell, comprising a means of measuring a relocation of anSMN complex component in a cell. In another embodiment, the presentinvention provides a kit for testing a modifier of p38 mitogen-activatedprotein kinase (MAPK) activity in a cell, comprising a means ofmeasuring a relocation of an SMN complex component in a cell. In anotherembodiment, a control cell is contacted with the protein synthesisinhibitor and the activator of p38 MAPK activity, but not the candidatecompound. In another embodiment, increase of the SMN complex componentin the nucleus and/or decrease in the cytoplasm is calculated relativeto the control cell. Each possibility represents a separate embodimentof the present invention.

In another embodiment, the present invention provides a system forscreening for a p38 MAPK modifier in a cell, comprising a kit forscreening a modifier of p38 mitogen-activated protein kinase (MAPK)activity in a cell, comprising a means of measuring a relocation of anSMN complex component in a cell. In another embodiment, the presentinvention provides a system for testing a p38 MAPK modifier in a cell,comprising a kit for testing a modifier of p38 mitogen-activated proteinkinase (MAPK) activity in a cell, comprising a means of measuring arelocation of an SMN complex component in a cell.

In one embodiment, “protein synthesis” comprises the multi-step processcomprising the translation of the genetic information encoded inmessenger RNAs (mRNAs) into proteins. In another embodiment, the methodof the present invention is based on cellular localization of thesurvival of motor neurons protein (SMN). In another embodiment, proteinsynthesis inhibitors cause the SMN complex (and several of itsassociated proteins, called Gemins) to relocalize from the cytoplasm tothe nucleus. In another embodiment, protein synthesis inhibitors causethe SMN associated proteins to relocalize from the cytoplasm to thenucleus. In another embodiment, SMN associated proteins are Gemins.

In another embodiment, the SMN complex component is a Gem. In anotherembodiment, Gems are discrete SMN complex bodies in the nucleus. Inanother embodiment, “an SMN complex component” comprise, in addition toSMN, other proteins termed “Gemins.”

In another embodiment, the Gemin protein is Gemin1 protein. In anotherembodiment, the sequence of the Gemin1 protein comprises the sequence:MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNGDICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIASIDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENSRSPGNKSDNIKPKSAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSCWLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRCSHSL N(SEQ. ID NO: 1). In another embodiment, the Gemin1 protein of thepresent invention comprises an amino acid sequence homologous to SEQ.ID. NO: 1. In another embodiment, the Gemin 1 protein is a Homo sapiensGemin1 protein. In another embodiment, the Gemin1 protein is from anon-human species. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the Gemin protein is Gemin2 protein. In anotherembodiment, the sequence of the Gemin2 protein comprises the sequence:MRRAELAGLKTMAWVPAESAVEELMPRLLPVEPCDLTEGFDPSVPPRTPQEYLRRVQIEAAQCPDVVVAQIDPKKLKRKQSVNISLSGCQPAPEGYSPTLQWQQQQVAQFSTVRQNVNKHRSHWKSQQLDSNVTMPKSEDEEGWKKFCLGEKLCADGAVGPATNESPGIDYVQIGFPPLLSIVSRMNQATVTSVLEYLSNWFGERDFTPELGRWLYALLACLEKPLLPEAHSLRQLARRCSEVRLLVDSKDDERVPALNLLICLVSRYFDQRDLADEPS (SEQ. ID NO:2). In another embodiment, the Gemin2 protein of the present inventioncomprises an amino acid sequence homologous to SEQ. ID. NO: 2. Inanother embodiment, the Gemin2 protein is a Homo sapiens Gemin2 protein.In another embodiment, the Gemin2 protein is from a non-human species.Each possibility represents a separate embodiment of the presentinvention.

In another embodiment, the Gemin protein is Gemin3 protein. In anotherembodiment, the sequence of the Gemin3 protein comprises the sequence:MAAAFEASGALAAVATAMPAEHVAVQVPAPEPTPGPVRILRTAQDLSSPRTRTGDVLLAEPADFESLLLSRPVLEGLRAAGFERPSPVQLKAIPLGRCGLDLIVQAKSGTGKTCVFSTIALDSLVLENLSTQILILAPTREIAVQIHSVITAIGIKMEGLECHVFIGGTPLSQDKTRLKKCHIAVGSPGRIKQLIELDYLNPGSIRLFILDEADKLLEEGSFQEQINWIYSSLPASKQMLAVSATYPEFLANALTKYMRDPTFVRLNSSDPSLIGLKQYYKVVNSYPLAHKVFEEKTQHLQELFSRIPFNQALVFSNLHSRAQHLADILSSKGFPAECISGNMNQNQRLDAMAKLKHFHCRVLISTDLTSRGIDAEKVNLVVNLDVPLDWETYMHRIGRAGRFGTLGLTVTYCCRGEEENMMMRIAQKCNINLLPLPDPIPSGLMEECVDWDVEVKAAVHTYGIASVPNQPLKKQIQKIERTLQIQKAHGDHMASSRNNSVSGLSVKSKNNTKQKLPVKSHSECGIIEKATSPKELGCDRQSEEQMKNSVQTPVENSTNSQHQVKEALPVSLPQIPCLSSFKIHQPYTLTFAELVEDYEHYIKEGLEKPVEIIRHYTGPGDQTVNPQNGFVRNKVIEQRVPVLASSSQSGDSESDSDSYSSRTSSQSKGNKSYLEGSSDNQLKDSESTPVDDRISLEQPPNGSDTPNPEKYQESPGIQMKTRLKEGASQRAKQSRRNLPRRSSFRLQTEAQEDDWYDCHREIRLSFSDTYQDYEEYWRAYYRAWQEYYAAASHSYYWNAQRHPSWMAAYHMNTIYLQE MMHSNQ(SEQ. ID NO: 3). In another embodiment, the Gemin3 protein of thepresent invention comprises an amino acid sequence homologous to SEQ.ID. NO: 3. In another embodiment, the Gemin3 protein is a Homo sapiensGemin3 protein. In another embodiment, the Gemin3 protein is from anon-human species. Each possibility represents a separate embodiment ofthe present invention.

In another embodiment, the Gemin protein is Gemin4 protein. In anotherembodiment, the sequence of the Gemin4 protein comprises the sequence:MDLGPLNICEEMTILHGGFLLAEQLFHPKALAELTKSDWERVGRPIVEALREISSAAAHSQPFAWKKKALIIIWAKVLQPHPVTPSDTETRWQEDLFFSVGNMIPTINHTILFELLKSLEASGLFIQLLMALPTTICHAELERFLEHVTVDTSAEDVAFFLDIWWEVMKHKGHPQDPLLSQFSAMAHKYLPALDEFPHPPKRLRSDPDACPTMPLLAMLLRGLTQIQSRILGPGRKCCALANLADMLTVFALTEDDPQEVSATVYLDKLATVISVWNSDTQNPYHQQALAEKVKEAERDVSLTSLAKLPSETIFVGCEFLHHLLREWGEELQAVLRSSQGTSYDSYRLCDSLTSFSQNATLYLNRTSLSKEDRQVVSELAECVRDFLRKTSTVLKNRALEDITASIAMAVIQQKMDRHMEVCYIFASEKKWAFSDEWVACLGSNRALFREPDLVLRLLETVIDVSTADRAIPESQIRQVIHLLECYADLSLPGKNKVLAGILRSWGRKGLSEKLLAYVEGFQEDLNTTFNQLTQSASEQGLAKAVASVARLVIVHPEVTVKKMCSLAVVNLGTHKFLAQILTAFPALRFVEVQGPNSSATFMVSCLKETVWMKFSTPKEEKQFLELLNCLMSPVKPQGIPVAALLEPDEVLKEFVLPFLRLDVEEVDLSLRIFIQTLEANACREEYWLQTCSPFPLLFSLCQLLDRFSKYWPLPKEKRCLSLDRKDLAIHILELLCEIVSANAETFSPDVWIKSLSWLHRKLEQLDWTVGLRLKSFFEGHFKCEVPATLFEICKLSEDEWTSQAHPGYGAGTGLLAWMECCCVSSGISERMLSLLVVDVGNPEEVRLFSKGFLVALVQVMPWCSPQEWQRLHQLTRRLLEKQLLHVPYSLEYIQFVPLLNLKPFAQELQLSVLFLRTFQFLCSHSCRNWLPLEGWNHVVKLLCGSLTRLLDSVRAIQAAGPWVQGPEQDLTQEALFVYTQVFCHALHIMAMLHPEVCEPLYVLALETLTCYETLSKTNPSVSSLLQRAHEQRFLKSIAEGIGPEERRQTLLQKMSSF (SEQ. ID NO: 4). In anotherembodiment, the Gemin4 protein of the present invention comprises anamino acid sequence homologous to SEQ. ID. NO: 4. In another embodiment,the Gemin4 protein is a Homo sapiens Gemin4 protein. In anotherembodiment, the Gemin4 protein is from a non-human species. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the Gemin protein is Gemin5 protein. In anotherembodiment, the sequence of the Gemin5 protein comprises the sequence:MGQEPRTLPPSPNWYCARCSDAVPGGLFGFAARTSVFLVRVGPGAGESPGTPPFRVIGELVGHTERVSGFTFSHHPGQYNLCATSSDDGTVKIWDVETKTVVTEHALHQHTISTLHWSPRVKDLIVSGDEKGVVFCYWFNRNDSQHLFIEPRTIFCLTCSPHHEDLVAIGYKDGIVVIIDISKKGEVIHRLRGHDDEIHSIAWCPLPGEDCLSINQEETSEEAEITNGNAVAQAPVTKGCYLATGSKDQTIRIWSCSRGRGVMILKLPFLKRRGGGIDPTVKERLWLTLHWPSNQPTQLVSSCFGGELLQWDLTQSWRRKYTLFSASSEGQNHSRIVFNLCPLQTEDDKQLLLSTSMDRDVKCWDIATLECSWTLPSLGGFAYSLAFSSVDIGSLAIGVGDGMIRVWNTLSIKNNYDVKNFWQGVKSKVTALCWHPTKEGCLAFGTDDGKVGLYDTYSNKPPQISSTYHKKTVYTLAWGPPVPPMSLGGEGDRPSLALYSCGGEGIVLQHNPWKLSGEAFDINKLIRDTNSIKYKLPVHTEISWKADGKIMALGNEDGSIEIFQIPNLKLICTIQQHHKLVNTISWHHEHGSQPELSYLMASGSNNAVIYVHNLKTVIESSPESPVTITEPYRTLSGHTAKITSVAWSPHHDGRLVSASYDGTAQVWDALREEPLCNFRGHRGRLLCVAWSPLDPDCIYSGADDFCVHKWLTSMQDHSRPPQGKKSIELEKKRLSQPKAKPKKKKKPTLRTPVKLESIDGNEEESMKENSGPVENGVSDQEGEEQAREPELPCGLAPAVSREPVICTPVSSGFEKSKVTINNKVILLKKEPPKEKPETLIKKRKARSLLPLSTSLDHRSKEELHQDCLVLATAKHSRELNEDVSADVEERFHLGLFTDRATLYRMIDIEGKGHLENGHPELFHQLMLWKGDLKGVLQTAAERGELTDNLVAMAPAAGYHVWLWAVEAFAKQLCFQDQYVKAASHLLSIHKVYEAVELLKSNHFYREAIAIAKARLRPEDPVLKDLYLSWGTVLERDGHYAVAAKCYLGATCAYDAAKVLAKKGDAASLRTAAELAAIVGEDELSASLALRCAQELLLANNWVGAQEALQLHESLQGQRLVFCLLELLSRHLEEKQLSEGKSSSSYHTWNTGTEGPFVERVTAVWKSIFSLDTPEQYQEAFQKLQNIKYPSATNNTPAKQLLLHICHDLTLAVLSQQMASWDEAVQALLRAVVRSYDSGSFTIMQEVYSAFLPDGCDHLRDKLGDHQSPATPAFKSLEAFFLYGRLYEFWWSLSRPCPNSSVWVRAGHRTLSVEPSQQLDTASTEETDPETSQPEPNRPSELDLRLTEEGERMLSTFKELFSEKHASLQNSQRTVAEVQETLAEMIRQHQKSQLCKSTANGPDKNEPEVEAEQPLCSSQSQCKEEKNEPLSLPELTKRLTEANQRMAKFPESIKAWPFPDVLECCLVLLLIRSHFPGCLAQEMQQQAQELLQKYGNTKTYRRHCQTFCM (SEQ. ID NO: 5). In anotherembodiment, the Gemin5 protein of the present invention comprises anamino acid sequence homologous to SEQ. ID. NO: 5. In another embodiment,the Gemin5 protein is a Homo sapiens Gemin5 protein. In anotherembodiment, the Gemin5 protein is from a non-human species. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the Gemin protein is Gemin6 protein. In anotherembodiment, the sequence of the Gemin6 protein comprises the sequence:MSEWMKKGPLEWQDYIYKEVRVTASEKNEYKGWVLTTDPVSANIVLVNFLEDGSMSVTGIMGHAVQTVETMNEGDHRVREKLMHLFTSGDCKAYSPEDLEERKNSLKKWLEKNHIPITEQGDAPRTLCVAGVLTIDPPYGPENCSSSNEIILSRVQDLIEGHLTASQ (SEQ. ID NO: 6). In another embodiment, the Gemin6 protein ofthe present invention comprises an amino acid sequence homologous toSEQ. ID. NO: 6. In another embodiment, the Gemin6 protein is a Homosapiens Gemin6 protein. In another embodiment, the Gemin6 protein isfrom a non-human species. Each possibility represents a separateembodiment of the present invention.

In another embodiment, the Gemin protein is Gemin7 protein. In anotherembodiment, the sequence of the Gemin7 protein comprises the sequence:MQTPVNIPVPVLRLPRGPDGFSRGFAPDGRRAPLRPEVPEIQECPIAQESLESQEQRARAALRERYLRSLLAMVGHQVSFTLHEGVRVAAHFGATDLDVANFYVSQLQTPIGVQAEALLRCSDIISYTFKP (SED. ID NO: 7). In anotherembodiment, the Gemin7 protein of the present invention comprises anamino acid sequence homologous to SEQ. ID. NO: 7. In another embodiment,the Gemin7 protein is a Homo sapiens Gemin7 protein. In anotherembodiment, the Gemin7 protein is from a non-human species. Eachpossibility represents a separate embodiment of the present invention.

In another embodiment, the Gemin protein of the present invention is amouse Gemin. In another embodiment, the Gemin protein of the presentinvention is a rat Gemin. In another embodiment, the Gemin protein ofthe present invention is a Drosophila melanogaster Gemin. In anotherembodiment, the Gemin protein of the present invention is a baboonGemin. In another embodiment, the Gemin protein of the present inventionis a guinea pig Gemin. In another embodiment, the Gemin protein of thepresent invention is a Drosophila melanogaster Gemin.

In another embodiment, the Gemin protein of the present invention is atleast 60% homologous to anyone SEQ. ID NOs: 1-7. In another embodiment,the Gemin protein of the present invention is at least 70% homologous toanyone SEQ. ID NOs: 1-7. In another embodiment, the Gemin protein of thepresent invention is at least 80% homologous to anyone SEQ. ID NOs: 1-7.In another embodiment, the Gemin protein of the present invention is atleast 90% homologous to anyone SEQ. ID NOs: 1-7. In another embodiment,the Gemin protein of the present invention is at least 95% homologous toanyone SEQ. ID NOs: 1-7.

In another embodiment, the SMN protein of the present inventionoligomerizes and forms a stable multiprotein complex that comprises ofSMN, Gemin2 (SIP1), Gemin3 (a DEAD-box RNA helicase), Gemin4, Gemin5 (aWD-repeat protein), Gemin6 and Gemin7. In another embodiment, the SMNprotein binds directly to Gemin 2, 3, 5 and 7, whereas Gemin 4 and 6require Gemin3 and 7, respectively, for interaction with SMN. In anotherembodiment, other proteins bind to the SMN protein. In anotherembodiment, these proteins, referred to as SMN complex substrates,include Sm and Sm-like (LSm) proteins, RNA helicase A, fibrillarin andGARI, the RNP proteins hnRNP U, hnRNP Q and hnRNP R, as well asp80-coilin, the protein marker for Cajal (coiled) bodies.

In another embodiment, protein synthesis inhibitors cause the SMNprotein and/or Gemins to relocalize from the cytoplasm to the nucleuswithin 1-48 hours. In another embodiment, protein synthesis inhibitorscause the SMN protein and/or Gemins to relocalize from the cytoplasm tothe nucleus within 1-3 hours. In another embodiment, protein synthesisinhibitors cause the SMN protein and/or Gemins to relocalize from thecytoplasm to the nucleus within 3-5 hours. In another embodiment,protein synthesis inhibitors cause the SMN protein and/or Gemins torelocalize from the cytoplasm to the nucleus within 5-7 hours. Inanother embodiment, protein synthesis inhibitors cause the SMN proteinand/or Gemins to relocalize from the cytoplasm to the nucleus within 7-9hours. In another embodiment, protein synthesis inhibitors cause the SMNprotein and/or Gemins to relocalize from the cytoplasm to the nucleuswithin 4-6 hours.

In another embodiment, protein synthesis inhibitors cause the SMNprotein and/or Gemins to relocalize from the cytoplasm to the nucleuswithin 9-12 hours. In another embodiment, protein synthesis inhibitorscause the SMN protein and/or Gemins to relocalize from the cytoplasm tothe nucleus within 12-15 hours. In another embodiment, protein synthesisinhibitors cause the SMN protein and/or Gemins to relocalize from thecytoplasm to the nucleus within 15-18 hours. In another embodiment,protein synthesis inhibitors cause the SMN protein and/or Gemins torelocalize from the cytoplasm to the nucleus within 18-21 hours. Inanother embodiment, protein synthesis inhibitors cause the SMN proteinand/or Gemins to relocalize from the cytoplasm to the nucleus within21-24 hours. In another embodiment, protein synthesis inhibitors causethe SMN protein and/or Gemins to relocalize from the cytoplasm to thenucleus within 24-30 hours. In another embodiment, protein synthesisinhibitors cause the SMN protein and/or Gemins to relocalize from thecytoplasm to the nucleus within 30-36 hours. In another embodiment,protein synthesis inhibitors cause the SMN protein and/or Gemins torelocalize from the cytoplasm to the nucleus within 36-42 hours. Inanother embodiment, protein synthesis inhibitors cause the SMN proteinand/or Gemins to relocalize from the cytoplasm to the nucleus within42-48 hours.

In another embodiment, protein synthesis inhibitors cause the SMNprotein and/or Gemins to relocalize from the cytoplasm to the nucleuswithin 1-8 hours in HeLa cells. In another embodiment, protein synthesisinhibitors cause the SMN protein and/or Gemins to relocalize from thecytoplasm to the nucleus within 2-7 hours in HeLa cells. In anotherembodiment, protein synthesis inhibitors cause the SMN protein and/orGemins to relocalize from the cytoplasm to the nucleus within 4-6 hoursin HeLa cells.

In another embodiment, the present invention provides a method forscreening for a p38 mitogen activated protein kinase (MAPK) modifier,comprising the steps of (a) contacting a cell with a candidate compoundand a p38 MAPK activator; and (b) calculating the level ofrelocalization of a SMN complex component from the cytoplasm to thenucleus. In another embodiment, the present invention provides a methodfor testing a p38 mitogen activated protein kinase (MAPK) modifier,comprising the steps of (a) contacting a cell with a candidate compoundand a p38 MAPK activator; and (b) calculating the level ofrelocalization of a SMN complex component from the cytoplasm to thenucleus.

In another embodiment, the present invention provides a method forscreening for a p38 MAPK modifier, comprising the steps of (a)contacting a cell with a candidate compound and an activator of JNK andp38 MAPK; and (b) calculating the level of relocalization of a SMNcomplex component from the cytoplasm to the nucleus. In anotherembodiment, the present invention provides a method for testing a p38MAPK modifier, comprising the steps of (a) contacting a cell with acandidate compound and an activator of JNK and p38 MAPK; and (b)calculating the level of relocalization of a SMN complex component fromthe cytoplasm to the nucleus.

In another embodiment, the present invention provides a method forscreening for a p38 MAPK modifier, comprising the steps of (a)contacting a cell with a candidate compound and a protein synthesisinhibitor; and (b) calculating the level of relocalization of a SMNcomplex component from the cytoplasm to the nucleus. In anotherembodiment, the present invention provides a method for testing a p38MAPK modifier, comprising the steps of (a) contacting a cell with acandidate compound and a protein synthesis inhibitor; and (b)calculating the level of relocalization of a SMN complex component fromthe cytoplasm to the nucleus.

In another embodiment, the present invention provides a method forscreening for a p38 MAPK modifier, comprising the steps of (a)contacting a cell with a candidate compound and anisomycin; and (b)calculating the level of relocalization of a SMN complex component fromthe cytoplasm to the nucleus. In another embodiment, the proteinsynthesis inhibitor is anisomycin. In another embodiment, the p38MAPK/JNK activator is anisomycin. In another embodiment, the presentinvention provides a method for testing a p38 MAPK modifier, comprisingthe steps of (a) contacting a cell with a candidate compound andanisomycin; and (b) calculating the level of relocalization of a SMNcomplex component from the cytoplasm to the nucleus. In anotherembodiment, the protein synthesis inhibitor is anisomycin. In anotherembodiment, the p38 MAPK/JNK activator is anisomycin.

In another embodiment, calculating a change in distribution of an SMNcomplex component in the cytoplasm and the nucleus of a cell comprisesmeasuring the level of a SMN complex component in the cytoplasm of saidcell, the nucleus of said cell, or a combination thereof. In anotherembodiment, calculating a change in distribution of an SMN complexcomponent in the cytoplasm and the nucleus of a cell comprises measuringthe distribution of an SMN complex component in the cytoplasm and thenucleus in a cell.

In another embodiment, the method of the present invention provides amethod for screening for a p38 MAPK, comprising the steps of contactinga cell with a candidate compound and a protein synthesis inhibitor andmeasuring an SMN complex component in the cytoplasm. In anotherembodiment, the method of the present invention provides a method fortesting a p38 MAPK, comprising the steps of contacting a cell with acandidate compound and a protein synthesis inhibitor and measuring anSMN complex component in the cytoplasm. In another embodiment, themethod of the present invention further provides setting a thresholdlevel of an SMN complex component in the cytoplasm in protein synthesisinhibitor treated cells. In another embodiment, the method of thepresent invention provides that contacting a cell with a proteinsynthesis inhibitor and a p38 MAPK inducer causes an incline in thelevels of an SMN complex component in the cytoplasm of the p38 MAPKinducer and protein synthesis inhibitor treated cells compared to cellstreated only with a protein synthesis inhibitor (FIG. 6).

In another embodiment, the method of the present invention provides amethod for screening for a p38 MAPK activator, comprising the steps ofcontacting a cell with a candidate compound and a protein synthesisinhibitor and measuring localization of an SMN complex component in thenucleus. In another embodiment, the method of the present inventionprovides a method for testing a p38 MAPK activator, comprising the stepsof contacting a cell with a candidate compound and a protein synthesisinhibitor and measuring localization of an SMN complex component in thenucleus. In another embodiment, the method of the present inventionprovides a method for screening for a p38 MAPK inhibitor, comprising thesteps of contacting a cell with a candidate compound and a proteinsynthesis inhibitor and measuring an SMN complex component in thenucleus. In another embodiment, the method of the present inventionfurther provides setting a threshold level of an SMN complex componentin the nucleus in protein synthesis inhibitor treated cells. In anotherembodiment, the method of the present invention provides that contactinga cell with a protein synthesis inhibitor and a p38 MAPK inducer causesa decline in the levels of an SMN complex component in the nucleus ofthe p38 MAPK inducer and protein synthesis inhibitor treated cellscompared to cells treated only with a protein synthesis inhibitor (FIG.6).

In another embodiment, the p38 MAPK of the present invention comprisesfour p38 MAPK isoforms. In another embodiment, the p38 MAPK of thepresent invention share at least 50% homology.

In another embodiment, the p38 MAPK of the present invention share atleast 60% homology. In another embodiment, the p38 MAPK of the presentinvention share at least 70% homology. In another embodiment, the p38MAPK of the present invention share at least 80% homology.

In another embodiment, the p38 MAPK of the present invention is a p38MAPK. In another embodiment, the p38 MAPK of the present invention is ap38h MAPK. In another embodiment, p38 MAPK and p38h MAPK areubiquitously expressed. In another embodiment, the p38 MAPK of thepresent invention is a p38g MAPK. In another embodiment, p38g MAPK ispredominantly expressed in skeletal muscles. In another embodiment, thep38 MAPK of the present invention is a p38h MAPK. In another embodiment,p38h MAPK is predominantly expressed in the lung, kidney, testis,pancreas, and small intestine. In another embodiment, the p38 MAPK ofthe present invention is activated by dual phosphorylation on Thr180 andTyr182 by upstream MAPK kinases such as MAP2K6 or MAP2K3 (MKK3/6), whichare activated by upstream MAPKKKs.

In another embodiment, the present invention provides a method forscreening for a p38 MAPK inhibitor comprising the steps of contacting acell with a candidate compound and anisomycin and measuring the level ofa SMN complex component in the cytoplasm of said cell, the nucleus ofsaid cell, or a combination thereof. In another embodiment, the presentinvention provides a method for testing a p38 MAPK inhibitor comprisingthe steps of contacting a cell with a candidate compound and anisomycinand measuring the level of a SMN complex component in the cytoplasm ofsaid cell, the nucleus of said cell, or a combination thereof.

In another embodiment, the method of the present invention comprises theuse of a compound that inhibits protein synthesis and activates MAPK. Inanother embodiment, the method of the present invention comprises theuse of a compound that inhibits protein synthesis and activates MAPK andJNK. In another embodiment, a compound that inhibits protein synthesisand activates MAPK and JNK will be used to screen p38 MAPK inhibitors.

In another embodiment, a candidate compound that induces SMN complexcomponents to accumulate in the cytoplasm of a treated cell, in thepresence of a compound possessing protein synthesis inhibition activityis scored as a MAPK inducer. In another embodiment, a candidate compoundthat induces SMN complex components to accumulate in the nucleus of atreated cell, in the presence of a compound possessing both proteinsynthesis inhibition activity and MAPK inducing activity is scored as aMAPK inhibitor. In another embodiment, a candidate compound that inducesSMN complex components to accumulate in the nucleus of a treated cell,in the presence of a compound possessing protein synthesis inhibitionactivity and an additional compound possessing MAPK inducing activity isscored as a MAPK inhibitor.

In another embodiment, p38 MAPK activators antagonize the effect ofprotein synthesis inhibitors causing SMN complex components torelocalize from the cytoplasm to the nucleus of a treated cell. Inanother embodiment, compounds or treatments that cause activation of p38MAPK, administered to cells at the same time the cells are treated withprotein synthesis inhibitor prevent the SMN protein from accumulating inthe nucleus. In another embodiment, p38 MAPK activators includeanisomycin and TNFα. In another embodiment, Anisomycin is both a proteinsynthesis inhibitor and an activator of p38 MAPK and JNK. In anotherembodiment, treatment of cells with anisomycin together with a p38 MAPKinhibitor such as SB202190 or SB203590 (but not with JNK inhibitors),cause SMN to accumulate in the nucleus. In another embodiment, themethod of the present invention comprises that cells are treated withanisomycin in each well and, in addition, with a library of compounds.In another embodiment, the library of compounds comprises smallmolecules. In another embodiment, the library of compounds comprisesproteins. In another embodiment, the library of compounds comprisessiRNA. In another embodiment, each compound is administered in adifferent well and the accumulation of SMN in the nucleus is monitored.

In another embodiment, the present invention provides that activityvalidations of a candidate compound of the present invention, itsspecific mechanism, and target are performed by additional assays thatare readily available to one of skill in the art.

In another embodiment, the methods of the present invention comprisescreening compounds that activate p38 MAPK. In another embodiment, cellsare treated with a protein synthesis inhibitor such as but not limitedto cycloheximide. In another embodiment, compounds that inhibit SMNaccumulation in the nucleus are scored as positive. In anotherembodiment, anisomycin and TNFα inhibit SMN accumulation in the nucleusin the presence of a protein synthesis inhibitor and are thus scored aspositive. In another embodiment, anisomycin and TNFα are activators ofp38 MAPK. In another embodiment, anisomycin and TNFα are antagonists ofthe protein synthesis inhibitor activity according to the methods of thepresent invention.

In another embodiment, cell-type specificity may be obtained by using avarious protein synthesis inhibitors such as but not limited toanisomycin, cycloheximide. In another embodiment, cell-type effectorspecificity may be obtained by using various p38 MAPK inducers such asbut not limited to TNFα, lipopolysaccharide (LPS), phenylephrine, orgonadotropin releasing hormone (GnRH).

In another, embodiment p38 modifiers identified by the methods of thepresent invention will be developed as drugs. In another, embodiment p38inhibitors identified by the methods of the present invention will bedeveloped as drugs for various clinical applications such as but notlimited to inflammatory diseases.

In another embodiment, in steady state SMN complex components arelocalized primarily in the cytoplasm. In another embodiment, inhibitionof protein synthesis causes SMN complex components to accumulate in thenucleus. In another embodiment, p38 MAPK inducers antagonize proteinsynthesis inhibitors effects on relocalization of SMN complexcomponents, causing SMN complex components to accumulate in thecytoplasm.

In another embodiment, Anisomycin causes SMN complex components toaccumulate in the cytoplasm of the treated cell. In another embodiment,Anisomycin inhibits protein synthesis. In another embodiment, Anisomycinis a p38 MAPK inducer. In another embodiment, Anisomycin antagonizesprotein synthesis inhibitors causing SMN to accumulate in the cytoplasm.In another embodiment, the p38 MAPK inducer activity of Anisomycin takescontrol over its protein synthesis inhibition activity thus causing SMNcomplex components to accumulate in the cytoplasm. In anotherembodiment, the p38 MAPK inducing activity of Anisomycin takes controlover another compound possessing protein synthesis inhibition activityand thus causing SMN complex components to accumulate in the cytoplasmof the treated cell.

In another embodiment, the methods of the present invention provide thatinhibition of Anisomycin's p38 MAPK inducing activity causes SMN complexcomponents to accumulate in the nucleus. In another embodiment,inhibition of Anisomycin's p38 MAPK inducing activity cause Anisomycinto possess only its protein inhibition activity which in turn causes SMNcomplex components to accumulate in the nucleus. In another embodiment,a p38 MAPK inhibitor inhibits Anisomycin's p38 MAPK inducing activity.In another embodiment, p38 MAPK inhibitors of the present inventioninclude SB202190, or SB203590.

In another embodiment, a p38 MAPK inhibitor of the present inventioninhibits Anisomycin's p38 MAPK inducer activity downstream to p38 MAPKactivation event.

In another embodiment, a p38 MAPK inducer and a p38 MAPK inhibitor arecompetitors. In another embodiment, a p38 MAPK inhibitor of the presentinvention has a stronger substrate affinity than the p38 MAPK inducerand thus antagonizes the p38 MAPK inducer. In another embodiment, a p38MAPK inducer has a stronger substrate affinity than the p38 MAPKinhibitor and thus antagonizes the p38 MAPK inhibitor.

In another embodiment, the methods of the present invention provide acompound comprising both p38 MAPK inducing activity and proteinsynthesis inhibition activity such as but not limited to Anisomycin. Inanother embodiment, a compound comprising both p38 MAPK inducingactivity and protein synthesis inhibition activity is used to identifycandidate compounds possessing a p38 MAPK inhibitor activity. In anotherembodiment, Anisomycin is used to identify candidate compoundspossessing a p38 MAPK inhibitor activity. In another embodiment,according to the methods of the present invention Anisomycin and anadditional compound possessing protein synthesis inhibition activity areused to identify candidate compounds possessing a p38 MAPK inhibitoractivity.

In another embodiment, the methods of the present invention provide atleast two compounds wherein one compound possesses a p38 MAPK inducingactivity and the other compound possesses protein synthesis inhibitionactivity. In another embodiment, the compound possessing a p38 MAPKinducing activity is selected from but not limited to TNFα orlipopolysaccharide (LPS). In another embodiment, the compound possessinga protein synthesis inhibition activity is selected from but not limitedto cycloheximide (CHX). In another embodiment, the methods of thepresent invention provide at least two compounds wherein one compoundpossesses a p38 MAPK inducing activity and the other compound possessesa protein synthesis inhibition activity. In another embodiment, thelatter two compounds are used to identify candidate compounds possessinga p38 MAPK inhibitor activity. In another embodiment, according to themethods of the present invention the two compounds are TNFα or LPS as ap38 MAPK inducer and cycloheximide as a protein synthesis inhibitor.

In another embodiment, according to the methods of the present inventionthe use of a compound possessing only a p38 MAPK inducing activityrequires the use of at least one additional compound possessing proteinsynthesis inhibition activity.

In another embodiment, the p38 MAPK inducer of the present invention isknown to a person of skill in the art. In another embodiment, the p38MAPK inhibitor of the present invention is known to a person of skill inthe art. In another embodiment, the protein synthesis inhibitor of thepresent invention is known to a person of skill in the art.

In another embodiment, the methods of the present invention provide thattreating cells with a compound possessing protein synthesis inhibitionactivity and a compound possessing p38 MAPK inducing activity results inan SMN complex component accumulation in the cytoplasm (FIG. 7). Inanother embodiment, the methods of the present invention provide thattreating cells with a compound possessing protein synthesis inhibitionactivity and a compound possessing both p38 MAPK inducing activity andprotein synthesis inhibition activity results in an SMN complexcomponent accumulation in the cytoplasm (FIG. 7). In another embodiment,the methods of the present invention provide that treating cells with acompound possessing p38 MAPK inhibition activity and a compoundpossessing both p38 MAPK inducing activity and protein synthesisinhibition activity results in an SMN complex component accumulation inthe nucleus (FIG. 7). In another embodiment, SMN complex componentsaccumulate in the cytoplasm when p38 MAPK is induced in the presence ofa protein synthesis inhibitor.

In another embodiment, the methods of the present invention providetesting an ability of a candidate compound to induce a p38 MAPKactivity, comprising the steps of contacting a cell with a proteinsynthesis inhibitor and said candidate compound. In another embodiment,the methods of the present invention provide that the ability of acandidate compound to induce a p38 MAPK is measured by the level ofrelocalization of an SMN complex component from the cytoplasm to thenucleus of the treated cell. In another embodiment, the methods of thepresent invention provide a method for screening a p38 MAPK activatorcomprising treating a cell with a protein synthesis inhibitor and acandidate compound. In another embodiment, the present inventionprovides that activation of p38 MAPK antagonizes protein synthesisinhibition with respect to an SMN complex component relocalization. Inanother embodiment, the present invention provides that activation ofp38 MAPK in the presence of a protein synthesis inhibitor results inaccumulation of an SMN complex component in the cytoplasm of the treatedcell.

In another embodiment, the candidate compound screened according to themethods of the present invention is applied in a concentration of 0.1μM-100 mM (example 7). In another embodiment, the candidate compoundscreened according to the methods of the present invention is applied ina concentration of 0.1-4 μM. In another embodiment, the candidatecompound screened according to the methods of the present invention isapplied in a concentration of 0.1-0.5 μM. In another embodiment, thecandidate compound screened according to the methods of the presentinvention is applied in a concentration of 1-2 V M. In anotherembodiment, the candidate compound screened according to the methods ofthe present invention is applied in a concentration of 2-3 μM. Inanother embodiment, the candidate compound screened according to themethods of the present invention is applied in a concentration of 3-5μM. In another embodiment, the candidate compound screened according tothe methods of the present invention is applied in a concentration of5-10 μM.

In another embodiment, the candidate compound screened according to themethods of the present invention is applied in a concentration of 10-100μM. In another embodiment, the candidate compound screened according tothe methods of the present invention is applied in a concentration of100 μM-1 mM. In another embodiment, the candidate compound screenedaccording to the methods of the present invention is applied in aconcentration of 1-5 mM. In another embodiment, the candidate compoundscreened according to the methods of the present invention is applied ina concentration of 5-15 mM. In another embodiment, the candidatecompound screened according to the methods of the present invention isapplied in a concentration of 15-30 mM. In another embodiment, thecandidate compound screened according to the methods of the presentinvention is applied in a concentration of 30-50 mM. In anotherembodiment, the candidate compound screened according to the methods ofthe present invention is applied in a concentration of 50-75 mM. Inanother embodiment, the candidate compound screened according to themethods of the present invention is applied in a concentration of 75-100mM.

In another embodiment, the method of the present invention is carriedout using cells cultured in miniaturized format. In another embodiment,cells cultured in miniaturized format of the present invention comprisemulti-well plates. In another embodiment, a multi-well plate of thepresent invention comprises 96 wells. In another embodiment, amulti-well plate of the present invention comprises 384 wells (example2). In another embodiment, a multi-well plate of the present inventioncomprises 1536 wells. In another embodiment, a multi-well plate of thepresent invention comprises from 2-5000 wells. In another embodiment, amulti-well plate of the present invention comprises from 20-3000 wells.In another embodiment, a multi-well plate of the present inventioncomprises from 96-2000 wells.

In another embodiment, the cells of the present invention are treatedfor several hours as indicated hereinabove in the presence of acandidate compound that potentially inhibits p38 MAPK (example 7). Inanother embodiment, the cells of the present invention are treated inthe presence of small molecules that potentially inhibit p38 MAPK. Inanother embodiment, the cells of the present invention are then fixed.In another embodiment, the cells of the present invention are furtherpermeabilized. In another embodiment, the cells of the present inventionare further contacted with an SMN specific ligand. In anotherembodiment, the SMN specific ligand labels an SMN complex component. Inanother embodiment, the SMN specific ligand labels SMC complexcomponent.

In another embodiment, the cells of the present invention are treatedfor several hours as indicated hereinabove in the presence of acandidate compound that potentially induces p38 MAPK (example 7). Inanother embodiment, the cells of the present invention are treated inthe presence of small molecules that potentially induce p38 MAPK. Inanother embodiment, the cells of the present invention are then fixed.In another embodiment, the cells of the present invention are furtherpermeabilized. In another embodiment, the cells of the present inventionare further contacted with an SMN specific ligand. In anotherembodiment, the SMN specific ligand labels an SMN complex component. Inanother embodiment, the SMN specific ligand labels SMC complexcomponent.

In another embodiment, the labeled SMN complex component is thendetected. In another embodiment, the labeled SMN complex component isthen detected by microscopy. In another embodiment, digital imagesrecord the microscopic images. In another embodiment, the digital imagesare collected from several fields in each well. In another embodiment,the microscopic images collected from several fields in each well arefurther analyzed by algorithmic imaging software. In another embodiment,the algorithmic imaging software monitors the relative amount of labeledSMN complex component in the nucleus. In another embodiment, the nucleusis defined by the signal of a nuclear specific stain. In anotherembodiment, the cytoplasm is defined by the signal of a cytoplasmicspecific stain. In another embodiment, the nucleus signal is collectedin a separate channel from the labeled SMN complex component (example2).

In another embodiment, the present invention provides method foridentifying p38 MAPK modifiers. In another embodiment, a method foridentifying modifiers of p38 MAPK can interdict enhanced, unregulated,p38 MAPK in disease. In another embodiment, the present inventionprovides that use of MAPK inhibitors identified by the methods of thepresent invention emerges as an attractive strategy because MAPKinhibitors are capable of reducing both the synthesis ofpro-inflammatory cytokines and their signaling.

In another embodiment, the present invention provides that use of MAPKinhibitors identified by the methods of the present invention can treata number of diseases, including rheumatoid arthritis, chronicinflammatory bowel diseases, neurodegenerative disorders, and septicshock.

In another embodiment, the present invention provides that use of MAPKinhibitors identified by the methods of the present invention inhibitinflammatory stimuli that activate macrophages. In another embodiment,MAPK inhibitors identified by the methods of the present inventioninhibit inflammatory stimuli derived from microbial products. In anotherembodiment, MAPK inhibitors identified by the methods of the presentinvention inhibit inflammatory stimuli derived from cytokines such asIL-1. In another embodiment, MAPK inhibitors identified by the methodsof the present invention inhibit inflammatory stimuli derived throughthe Toll receptors, IL-1 receptor (TIR) family or the TNF receptorfamily. In another embodiment, MAPK inhibitors identified by the methodsof the present invention inhibit inflammatory stimuli derived fromNF-kB. In another embodiment, MAPK inhibitors identified by the methodsof the present invention inhibit inflammatory stimuli derived from TNFα.In another embodiment, MAPK inhibitors identified by the methods of thepresent invention inhibit inflammatory stimuli derivedLipopolysaccharide (LPS), a component of bacterial wall.

In another embodiment, MAPK inhibitors identified by the methods of thepresent invention inhibit p38 MAPK activity upstream to p38 MAPK. Inanother embodiment, MAPK inhibitors identified by the methods of thepresent invention inhibit p38 MAPK activity downstream to p38 MAPK. Inanother embodiment, MAPK inducers identified by the methods of thepresent invention induce p38 MAPK activity upstream to p38 MAPK. Inanother embodiment, MAPK inducers identified by the methods of thepresent invention induce p38 MAPK activity downstream to p38 MAPK.

In another embodiment, protein synthesis inhibitors of the presentinvention are inhibitors of protein elongation. In another embodiment,inhibitors of protein elongation have direct consequence of proteinsynthesis inhibition. In another embodiment, inhibitors of proteinsynthesis of the present invention target ABL protein. In anotherembodiment, inhibitors of protein synthesis of the present inventiontarget PDGFR protein. In another embodiment, inhibitors of proteinsynthesis of the present invention target KIT protein.

In another embodiment, candidate p38 modifiers of the present inventionare screened on a primary cell culture derived from a tumor. In anotherembodiment, candidate p38 modifiers of the present invention arescreened on a primary cell culture derived from a cancer patientsuffering from one of the cancers listed hereinabove. In anotherembodiment, candidate p38 modifiers of the present invention arescreened on a cancer cell line. In another embodiment, candidate p38modifiers of the present invention are screened on a cancer cell line ofa hematopoietic lineage. In another embodiment, cancer is associatedwith elevated p38 mitogen-activated protein kinase (MAPK) enzymaticactivity in cancerous cells. In another embodiment, cancer ischaracterized by elevated p38 mitogen-activated protein kinase (MAPK)enzymatic activity in cancerous cells.

In another embodiment, the present invention provides a method oftesting a candidate compound for an ability to induce p38 MAPK dependentapoptosis, comprising the steps of contacting a cell with a candidatecompound and an additional compound, wherein the additional compound iscapable of inhibiting protein synthesis in a cell and increasing anenzymatic activity of p38 MAPK in a cell; and measuring the level of aSMN complex component in the cytoplasm of a cell, the nucleus of a cell,or a combination thereof, whereby, if a candidate compound increases theamount of the SMN complex component in the nucleus, decreases the amountof the SMN complex component in the cytoplasm, or a combination thereof,then the compound exhibits an ability to decrease MAPK enzymaticactivity. In another embodiment, the present invention further providesdetection of pro-apoptotic cell derived compounds (e.g. hormones, growthfactors, nitric oxide, or cytokines) that are measured according tomethods known to one of skill in the art. In another embodiment,inhibition of p38 MAPK enzymatic activity is measured according to themethods of the present invention in cells secreting pro-apoptoticcompounds. In another embodiment, induction of p38 MAPK enzymaticactivity is measured according to the methods of the present inventionin cells secreting pro-apoptotic compounds. In another embodiment,inhibition of p38 MAPK enzymatic activity is measured according to themethods of the present invention in cells undergoing apoptosis. Inanother embodiment, induction of p38 MAPK enzymatic activity is measuredaccording to the methods of the present invention in cells undergoingapoptosis.

In another embodiment, ability to decrease MAPK enzymatic activitycorrelates with ability to induce the expression of pro-apoptoticcytokines or hormones in a target cell. In another embodiment, abilityto decrease MAPK enzymatic activity correlates with ability to inducethe pro-apoptotic oxidative stress in a target cell. In anotherembodiment, ability to inhibit MAPK enzymatic activity correlates withability to induce the expression of pro-apoptotic cytokines or hormonesin a target cell. In another embodiment, ability to induce MAPKenzymatic activity correlates with ability to induce the expression ofpro-apoptotic cytokines or hormones in a target cell. In anotherembodiment, ability to increase MAPK enzymatic activity correlates withability to induce the expression of pro-apoptotic cytokines or hormonesin a target cell. In another embodiment, ability to increase MAPKenzymatic activity correlates with ability to induce pro-apoptoticoxidative stress in a target cell. In another embodiment, ability toincrease MAPK enzymatic activity correlates with ability to inducepro-apoptotic nitric oxide (NO) stress in a target cell.

In another embodiment, induction of apoptosis by a candidate compound ofthe present invention correlates with a steady state level of MAPKenzymatic activity. In another embodiment, induction of expression ofpro-apoptotic cytokines in a target cell by a candidate compound of thepresent invention correlates with a steady state level of MAPK enzymaticactivity. In another embodiment, inhibition of apoptosis by a candidatecompound of the present invention correlates with a steady state levelof MAPK enzymatic activity. In another embodiment, inhibition ofexpression of pro-apoptotic cytokines or hormones in a target cell by acandidate compound of the present invention correlates with a steadystate level of MAPK enzymatic activity. In another embodiment,inhibition of pro-apoptotic NO in a target cell by a candidate compoundof the present invention correlates with a steady state level of MAPKenzymatic activity.

In another embodiment, the present invention provides a method oftesting a candidate compound for an ability to induce p38 MAPK dependentnecrosis, comprising the steps of contacting a cell with a candidatecompound and an additional compound, wherein the additional compound iscapable of inhibiting protein synthesis in a cell and increasing anenzymatic activity of p38 MAPK in a cell; and measuring the level of aSMN complex component in the cytoplasm of a cell, the nucleus of a cell,or a combination thereof, whereby, if a candidate compound increases theamount of the SMN complex component in the nucleus, decreases the amountof the SMN complex component in the cytoplasm, or a combination thereof,then the compound exhibits an ability to decrease MAPK enzymaticactivity. In another embodiment, the present invention further providesdetection of pro-necrotic cell derived compounds (e.g. hormones, growthfactors, nitric oxide, or cytokines) that are measured according tomethods known to one of skill in the art. In another embodiment,inhibition of p38 MAPK enzymatic activity is measured according to themethods of the present invention in cells undergoing necrosis. Inanother embodiment, induction of p38 MAPK enzymatic activity is measuredaccording to the methods of the present invention in cells undergoingnecrosis.

In another embodiment, ability to decrease MAPK enzymatic activitycorrelates with ability to induce stress in a target cell. In anotherembodiment, ability to decrease MAPK enzymatic activity correlates withability to induce oxidative stress in a target cell. In anotherembodiment, ability to decrease MAPK enzymatic activity correlates withability to induce hypoxia in a target cell. In another embodiment,ability to decrease MAPK enzymatic activity correlates with starving atarget cell. In another embodiment, ability to decrease MAPK enzymaticactivity correlates with ability to induce necrosis in a target cell. Inanother embodiment, ability to inhibit MAPK enzymatic activitycorrelates with ability to induce necrosis in a target cell. In anotherembodiment, ability to increase MAPK enzymatic activity correlates withability to induce stress in a target cell. In another embodiment,ability to increase MAPK enzymatic activity correlates with ability toinduce oxidative stress in a target cell. In another embodiment, abilityto increase MAPK enzymatic activity correlates with ability to inducehypoxia in a target cell. In another embodiment, ability to increaseMAPK enzymatic activity correlates with starving a target cell. Inanother embodiment, ability to increase MAPK enzymatic activitycorrelates with ability to induce necrosis in a target cell.

In another embodiment, induction of necrosis by a candidate compound ofthe present invention correlates with a steady state level of MAPKenzymatic activity. In another embodiment, induction of expression ofpro-necrotic cytokines in a target cell by a candidate compound of thepresent invention correlates with a steady state level of MAPK enzymaticactivity. In another embodiment, inhibition of necrosis by a candidatecompound of the present invention correlates with a steady state levelof MAPK enzymatic activity. In another embodiment, inhibition ofexpression of pro-necrotic cytokines or hormones in a target cell by acandidate compound of the present invention correlates with a steadystate level of MAPK enzymatic activity.

In another embodiment, the present invention provides a method oftesting a candidate compound for an ability to induce p38 MAPK dependentinflammation, comprising the steps of contacting a cell with a candidatecompound and an additional compound, wherein the additional compound iscapable of inhibiting protein synthesis in a cell and increasing anenzymatic activity of p38 MAPK in a cell; and measuring the level of aSMN complex component in the cytoplasm of a cell, the nucleus of a cell,or a combination thereof, whereby, if a candidate compound increases theamount of the SMN complex component in the nucleus, decreases the amountof the SMN complex component in the cytoplasm, or a combination thereof,then the compound exhibits an ability to decrease MAPK enzymaticactivity. In another embodiment, the present invention further providesdetection of pro-inflammatory cell derived compounds (e.g. IL-1, IL-6,TNF-α, and TGF-β) that are measured according to methods known to one ofskill in the art. In another embodiment, inhibition of p38 MAPKenzymatic activity is measured according to the methods of the presentinvention in cells secreting pro-inflammatory compounds. In anotherembodiment, induction of p38 MAPK enzymatic activity is measuredaccording to the methods of the present invention in cells secretingpro-inflammatory compounds. In another embodiment, ability to decreaseMAPK enzymatic activity correlates with ability to induce the expressionof pro-inflammatory cytokines in a target cell. In another embodiment,ability to inhibit MAPK enzymatic activity correlates with ability toinduce the expression of pro-inflammatory cytokines in a target cell. Inanother embodiment, ability to induce MAPK enzymatic activity correlateswith ability to induce the expression of pro-inflammatory cytokines in atarget cell. In another embodiment, ability to increase MAPK enzymaticactivity correlates with ability to induce the expression ofpro-inflammatory cytokines in a target cell.

In another embodiment, induction of inflammation by a candidate compoundof the present invention correlates with a steady state level of MAPKenzymatic activity. In another embodiment, induction of expression ofpro-inflammatory cytokines in a target cell by a candidate compound ofthe present invention correlates with a steady state level of MAPKenzymatic activity. In another embodiment, inhibition of inflammation bya candidate compound of the present invention correlates with a steadystate level of MAPK enzymatic activity. In another embodiment,inhibition of expression of pro-inflammatory cytokines in a target cellby a candidate compound of the present invention correlates with asteady state level of MAPK enzymatic activity.

In another embodiment, the methods of the present invention arepreformed on a cell. In another embodiment, the cell of the presentinvention is a eukaryotic cell. In another embodiment, the cell of thepresent invention is an epidermal keratinocyte. In another embodiment,the cell of the present invention is an epidermal basal cell. In anotherembodiment, the cell of the present invention is a keratinocyte offingernails or toenails. In another embodiment, the cell of the presentinvention is a nail bed basal cell. In another embodiment, the cell ofthe present invention is a stem cell. In another embodiment, the cell ofthe present invention is a medullary hair shaft cell. In anotherembodiment, the cell of the present invention is a cortical hair shaftcell. In another embodiment, the cell of the present invention is acuticular hair shaft cell. In another embodiment, the cell of thepresent invention is a cuticular hair root sheath cell. In anotherembodiment, the cell of the present invention is a hair root sheath cellof Huxley's layer. In another embodiment, the cell of the presentinvention is a hair root sheath cell of Henle's layer. In anotherembodiment, the cell of the present invention is an external hair rootsheath cell. In another embodiment, the cell of the present invention isa hair matrix cell. In another embodiment, the cell of the presentinvention is a prokaryotic cell.

In another embodiment, the cell of the present invention is a wetstratified barrier epithelial cell. In another embodiment, the cell ofthe present invention is a surface epithelial cell of stratifiedsquamous epithelium of the cornea. In another embodiment, the cell ofthe present invention is a surface epithelial cell of stratifiedsquamous epithelium of the tongue. In another embodiment, the cell ofthe present invention is a surface epithelial cell of stratifiedsquamous epithelium of the oral cavity. In another embodiment, the cellof the present invention is a surface epithelial cell of stratifiedsquamous epithelium of the esophagus. In another embodiment, the cell ofthe present invention is a surface epithelial cell of stratifiedsquamous epithelium of the anal canal. In another embodiment, the cellof the present invention is a surface epithelial cell of stratifiedsquamous epithelium of the distal urethra. In another embodiment, thecell of the present invention is a surface epithelial cell of stratifiedsquamous epithelium of the vagina.

In another embodiment, the cell of the present invention is a basal cellof epithelia of the cornea. In another embodiment, the cell of thepresent invention is a basal cell of epithelia of the tongue. In anotherembodiment, the cell of the present invention is a basal cell ofepithelia of the oral cavity. In another embodiment, the cell of thepresent invention is a basal cell of epithelia of the esophagus. Inanother embodiment, the cell of the present invention is a basal cell ofepithelia of the anal canal. In another embodiment, the cell of thepresent invention is a basal cell of epithelia of the distal urethra. Inanother embodiment, the cell of the present invention is a basal cell ofepithelia of the vagina.

In another embodiment, the cell of the present invention is a urinaryepithelium cell. In another embodiment, the cell of the presentinvention is an exocrine secretory epithelial cell. In anotherembodiment, the cell of the present invention is a salivary gland mucouscell. In another embodiment, the cell of the present invention is asalivary gland serous cell. In another embodiment, the cell of thepresent invention is a Von Ebner's gland cell. In another embodiment,the cell of the present invention is a mammary gland cell. In anotherembodiment, the cell of the present invention is a lacrimal gland cell.In another embodiment, the cell of the present invention is a ceruminousgland cell. In another embodiment, the cell of the present invention isan eccrine sweat gland dark cell. In another embodiment, the cell of thepresent invention is an eccrine sweat gland clear cell. In anotherembodiment, the cell of the present invention is an apocrine sweat glandcell. In another embodiment, the cell of the present invention is agland of moll cell. In another embodiment, the cell of the presentinvention is a sebaceous gland cell. In another embodiment, the cell ofthe present invention is a Bowman's gland cell. In another embodiment,the cell of the present invention is a Brunner's gland cell. In anotherembodiment, the cell of the present invention is a seminal vesicle cell.In another embodiment, the cell of the present invention is a prostategland cell. In another embodiment, the cell of the present invention isa bulbourethral gland cell. In another embodiment, the cell of thepresent invention is a Bartholin's gland cell. In another embodiment,the cell of the present invention is a gland of Littre cell. In anotherembodiment, the cell of the present invention is a uterus endometriumcell. In another embodiment, the cell of the present invention is agoblet cell. In another embodiment, the cell of the present invention isa stomach lining mucous cell.

In another embodiment, the cell of the present invention is a gastricgland cell. In another embodiment, the cell of the present invention isa gastric gland zymogenic cell. In another embodiment, the cell of thepresent invention is a gastric gland oxyntic cell. In anotherembodiment, the cell of the present invention is a pancreatic cell. Inanother embodiment, the cell of the present invention is a pancreaticacinar cell. In another embodiment, the cell of the present invention isa paneth cell. In another embodiment, the cell of the present inventionis a pneumocyte. In another embodiment, the cell of the presentinvention is a Clara cell of lung.

In another embodiment, the cell of the present invention is a hormonesecreting cell. In another embodiment, the cell of the present inventionis an anterior pituitary cell. In another embodiment, the cell of thepresent invention is a somatotrope. In another embodiment, the cell ofthe present invention is a lactotrope. In another embodiment, the cellof the present invention is a thyrotrope. In another embodiment, thecell of the present invention is a gonadotrope. In another embodiment,the cell of the present invention is a corticotrope. In anotherembodiment, the cell of the present invention is an intermediatepituitary cell. In another embodiment, the cell of the present inventionis a magnocellular neurosecretory cell. In another embodiment, the cellof the present invention is an oxytocin secreting cell. In anotherembodiment, the cell of the present invention is a serotonin secretingcell. In another embodiment, the cell of the present invention is anendorphin secreting cell. In another embodiment, the cell of the presentinvention is a somatostatin secreting cell. In another embodiment, thecell of the present invention is a gastrin secreting cell. In anotherembodiment, the cell of the present invention is a secretin secretingcell. In another embodiment, the cell of the present invention is acholecystokinin secreting cell. In another embodiment, the cell of thepresent invention is an insulin secreting cell. In another embodiment,the cell of the present invention is a glucagon secreting cell. Inanother embodiment, the cell of the present invention is a bombesinsecreting cell. In another embodiment, the cell of the present inventionis a thyroid gland cell. In another embodiment, the cell of the presentinvention is a thyroid epithelial cell. In another embodiment, the cellof the present invention is a parafollicular cell. In anotherembodiment, the cell of the present invention is a parathyroid glandcell. In another embodiment, the cell of the present invention is aparathyroid chief cell. In another embodiment, the cell of the presentinvention is an oxyphil cell.

In another embodiment, the cell of the present invention is an adrenalgland cell. In another embodiment, the cell of the present invention isa chromaffin cell. In another embodiment, the cell of the presentinvention is a steroid hormones secreting cell. In another embodiment,the cell of the present invention is a Leydig cell. In anotherembodiment, the cell of the present invention is a theca interna cell.In another embodiment, the cell of the present invention is a corpusluteum cell. In another embodiment, the cell of the present invention isa kidney j uxtaglomerular apparatus cell. In another embodiment, thecell of the present invention is a macula densa cell. In anotherembodiment, the cell of the present invention is a peripolar cell. Inanother embodiment, the cell of the present invention is a mesangialcell. In another embodiment, the cell of the present invention is anintestinal brush border cell. In another embodiment, the cell of thepresent invention is an exocrine gland striated duct cell. In anotherembodiment, the cell of the present invention is a gall bladderepithelial cell. In another embodiment, the cell of the presentinvention is a kidney proximal tubule brush border cell. In anotherembodiment, the cell of the present invention is a kidney distal tubulecell. In another embodiment, the cell of the present invention is aductulus efferens nonciliated cell. In another embodiment, the cell ofthe present invention is an epididymal principal cell. In anotherembodiment, the cell of the present invention is an epididymal basalcell.

In another embodiment, the cell of the present invention is a storagecell. In another embodiment, the cell of the present invention is ahepatocyte. In another embodiment, the cell of the present invention isa white fat cell. In another embodiment, the cell of the presentinvention is a brown fat cell. In another embodiment, the cell of thepresent invention is a liver lipocyte.

In another embodiment, the cell of the present invention is a barrierfunction cell. In another embodiment, the cell of the present inventionis a type I pneumocyte. In another embodiment, the cell of the presentinvention is a pancreatic duct cell. In another embodiment, the cell ofthe present invention is a nonstriated duct cell. In another embodiment,the cell of the present invention is a kidney glomerulus parietal cell.In another embodiment, the cell of the present invention is a kidneyglomerulus podocyte. In another embodiment, the cell of the presentinvention is a loop of Henle thin segment cell. In another embodiment,the cell of the present invention is a kidney collecting duct cell. Inanother embodiment, the cell of the present invention is a duct cell. Inanother embodiment, the cell of the present invention is an epithelialcell lining closed internal body cavity. In another embodiment, the cellof the present invention is a blood vessel cell. In another embodiment,the cell of the present invention is a lymphatic vascular endothelialfenestrated cell. In another embodiment, the cell of the presentinvention is a blood vessel or lymphatic vascular endothelial continuouscell. In another embodiment, the cell of the present invention is ablood vessel or lymphatic vascular endothelial splenic cell. In anotherembodiment, the cell of the present invention is a synovial cell. Inanother embodiment, the cell of the present invention is a serosal cell.In another embodiment, the cell of the present invention is a squamouscell. In another embodiment, the cell of the present invention is acolumnar cell of endolymphatic sac with microvilli. In anotherembodiment, the cell of the present invention is a columnar cell ofendolymphatic sac without microvilli. In another embodiment, the cell ofthe present invention is a dark cell. In another embodiment, the cell ofthe present invention is a vestibular membrane cell. In anotherembodiment, the cell of the present invention is a stria vascularisbasal cell. In another embodiment, the cell of the present invention isa stria vascularis marginal cell. In another embodiment, the cell of thepresent invention is a cell of Claudius. In another embodiment, the cellof the present invention is a cell of Boettcher. In another embodiment,the cell of the present invention is a choroid plexus cell. In anotherembodiment, the cell of the present invention is a pia-arachnoidsquamous cell. In another embodiment, the cell of the present inventionis a pigmented ciliary epithelium cell. In another embodiment, the cellof the present invention is a nonpigmented ciliary epithelium cell. Inanother embodiment, the cell of the present invention is a cornealendothelial cell. In another embodiment, the cell of the presentinvention is a ciliated cell with propulsive function. In anotherembodiment, the cell of the present invention is a respiratory tractciliated cell. In another embodiment, the cell of the present inventionis an oviduct ciliated cell. In another embodiment, the cell of thepresent invention is a uterine endometrial ciliated cell. In anotherembodiment, the cell of the present invention is a rete testis cilatedcell. In another embodiment, the cell of the present invention is aductulus efferens ciliated cell. In another embodiment, the cell of thepresent invention is a ciliated ependymal cell of central nervoussystem.

In another embodiment, the cell of the present invention is anextracellular matrix secretion cell. In another embodiment, the cell ofthe present invention is an ameloblast epithelial cell. In anotherembodiment, the cell of the present invention is a planum semilunatumepithelial cell. In another embodiment, the cell of the presentinvention is an organ of Corti interdental epithelial cell. In anotherembodiment, the cell of the present invention is a fibroblast. Inanother embodiment, the cell of the present invention is a looseconnective tissue fibroblast. In another embodiment, the cell of thepresent invention is a corneal fibroblast. In another embodiment, thecell of the present invention is a tendon fibroblast. In anotherembodiment, the cell of the present invention is a bone marrow reticulartissue fibroblast. In another embodiment, the cell of the presentinvention is a nonepithelial fibroblast. In another embodiment, the cellof the present invention is a pericyte. In another embodiment, the cellof the present invention is a nucleus pulposus cell of intervertebraldisc. In another embodiment, the cell of the present invention is acementoblast. In another embodiment, the cell of the present inventionis a cementocyte. In another embodiment, the cell of the presentinvention is an odontoblast. In another embodiment, the cell of thepresent invention is an odontocyte.

In another embodiment, the cell of the present invention is achondrocyte. In another embodiment, the cell of the present invention isa hyaline cartilage chondrocyte. In another embodiment, the cell of thepresent invention is a fibrocartilage chondrocyte. In anotherembodiment, the cell of the present invention is an elastic cartilagechondrocyte. In another embodiment, the cell of the present invention isan osteoblast. In another embodiment, the cell of the present inventionis an osteocyte. In another embodiment, the cell of the presentinvention is an osteoprogenitor cell. In another embodiment, the cell ofthe present invention is a hyalocyte. In another embodiment, the cell ofthe present invention is a stellate cell. In another embodiment, thecell of the present invention is a contractile cell.

In another embodiment, the cell of the present invention is a musclecell. In another embodiment, the cell of the present invention is a redskeletal muscle cell. In another embodiment, the cell of the presentinvention is a white skeletal muscle cell. In another embodiment, thecell of the present invention is an intermediate skeletal muscle cell.In another embodiment, the cell of the present invention is a nuclearbag cell. In another embodiment, the cell of the present invention is anuclear chain cell. In another embodiment, the cell of the presentinvention is a satellite cell. In another embodiment, the cell of thepresent invention is a heart muscle cell. In another embodiment, thecell of the present invention is a nodal heart muscle cell. In anotherembodiment, the cell of the present invention is a purkinje fiber cell.In another embodiment, the cell of the present invention is a smoothmuscle cell. In another embodiment, the cell of the present invention isa myoepithelial cell.

In another embodiment, the cell of the present invention is a bloodcell. In another embodiment, the cell of the present invention is animmune system cell. In another embodiment, the cell of the presentinvention is a red blood cell. In another embodiment, the cell of thepresent invention is a megakaryocyte.

In another embodiment, the cell of the present invention is a monocyte.In another embodiment, the cell of the present invention is macrophage.In another embodiment, the cell of the present invention is an epidermalLangerhans cell. In another embodiment, the cell of the presentinvention is an osteoclast. In another embodiment, the cell of thepresent invention is a dendritic cell. In another embodiment, the cellof the present invention is a microglial cell. In another embodiment,the cell of the present invention is a neutrophil. In anotherembodiment, the cell of the present invention is an eosinophil. Inanother embodiment, the cell of the present invention is a basophile. Inanother embodiment, the cell of the present invention is a mast cell.

In another embodiment, the cell of the present invention is a T-Helpercell. In another embodiment, the cell of the present invention is aT-suppressor cell. In another embodiment, the cell of the presentinvention is a cytotoxic T cell. In another embodiment, the cell of thepresent invention is a B cell. In another embodiment, the cell of thepresent invention is a natural killer cell. In another embodiment, thecell of the present invention is a reticulocyte. In another embodiment,the cell of the present invention is a stem cell. In another embodiment,the cell of the present invention is a committed progenitor for theblood and immune system.

In another embodiment, the cell of the present invention is a sensorytransducer cell. In another embodiment, the cell of the presentinvention is an auditory inner hair cell of organ of Corti. In anotherembodiment, the cell of the present invention is an auditory outer haircell of organ of Corti. In another embodiment, the cell of the presentinvention is a basal olfactory epithelium cell. In another embodiment,the cell of the present invention is a cold-sensitive primary sensoryneuron. In another embodiment, the cell of the present invention is aheat-sensitive primary sensory neuron. In another embodiment, the cellof the present invention is a merkel cell. In another embodiment, thecell of the present invention is an olfactory receptor neuron. Inanother embodiment, the cell of the present invention is apain-sensitive primary sensory neuron. In another embodiment, the cellof the present invention is a photoreceptor rod cell. In anotherembodiment, the cell of the present invention is a photoreceptorblue-sensitive cone cell. In another embodiment, the cell of the presentinvention is a photoreceptor green-sensitive cone cell. In anotherembodiment, the cell of the present invention is a photoreceptorred-sensitive cone cell. In another embodiment, the cell of the presentinvention is a proprioceptive primary sensory neuron. In anotherembodiment, the cell of the present invention is a touch-sensitiveprimary sensory neuron. In another embodiment, the cell of the presentinvention is a type I carotid body cell. In another embodiment, the cellof the present invention is a type II carotid body cell. In anotherembodiment, the cell of the present invention is a type I hair cell ofvestibular apparatus. In another embodiment, the cell of the presentinvention is a type II hair cell of vestibular apparatus. In anotherembodiment, the cell of the present invention is a type I taste budcell. In another embodiment, the cell of the present invention is anautonomic neuron cell. In another embodiment, the cell of the presentinvention is a cholinergic neural cell. In another embodiment, the cellof the present invention is an adrenergic neural cell. In anotherembodiment, the cell of the present invention is a peptidergic neuralcell. In another embodiment, the cell of the present invention is asense organ or peripheral neuron supporting cell. In another embodiment,the cell of the present invention is an inner pillar cell of organ ofCorti. In another embodiment, the cell of the present invention is anouter pillar cell of organ of Corti. In another embodiment, the cell ofthe present invention is an inner phalangeal cell of organ of Corti. Inanother embodiment, the cell of the present invention is an outerphalangeal cell of organ of Corti. In another embodiment, the cell ofthe present invention is a border cell of organ of Corti. In anotherembodiment, the cell of the present invention is a Hensen cell of organof Corti. In another embodiment, the cell of the present invention is avestibular apparatus supporting cell. In another embodiment, the cell ofthe present invention is a type I taste bud supporting cell. In anotherembodiment, the cell of the present invention is an olfactory epitheliumsupporting cell. In another embodiment, the cell of the presentinvention is a Schwann cell. In another embodiment, the cell of thepresent invention is a satellite cell encapsulating peripheral nervecell bodies. In another embodiment, the cell of the present invention isan enteric glial cell.

In another embodiment, the cell of the present invention is a centralnervous system neuron. In another embodiment, the cell of the presentinvention is a glial cell. In another embodiment, the cell of thepresent invention is an astrocyte. In another embodiment, the cell ofthe present invention is an oligodendrocyte. In another embodiment, thecell of the present invention is a spindle neuron. In anotherembodiment, the cell of the present invention is a lens cell. In anotherembodiment, the cell of the present invention is an anterior lensepithelial cell. In another embodiment, the cell of the presentinvention is a crystalline-containing lens fiber cell. In anotherembodiment, the cell of the present invention is a karan cell. Inanother embodiment, the cell of the present invention is a pigment cell.In another embodiment, the cell of the present invention is amelanocyte. In another embodiment, the cell of the present invention isa retinal pigmented epithelial cell.

In another embodiment, the cell of the present invention is a germ cell.In another embodiment, the cell of the present invention is an oogonium.In another embodiment, the cell of the present invention is an oocyte.In another embodiment, the cell of the present invention is a spermatid.In another embodiment, the cell of the present invention is aspermatocyte. In another embodiment, the cell of the present inventionis a spermatogonium cell. In another embodiment, the cell of the presentinvention is a spermatozoon.

In another embodiment, the cell of the present invention is a nursecell. In another embodiment, the cell of the present invention is anovarian follicle cell. In another embodiment, the cell of the presentinvention is a sertoli cell. In another embodiment, the cell of thepresent invention is a thymus epithelial cell.

In another embodiment, the cell of the present invention is derived froman organ. In another embodiment, the cell of the present invention isderived from a tissue. In another embodiment, the cell of the presentinvention is derived from a cell line. In another embodiment, the cellof the present invention is derived from a primary cell culture. Inanother embodiment, the cell of the present invention is a HeLa cell. Inanother embodiment, the cell of the present invention is a U2OS cell.

In another embodiment, the cell of the present invention is a plantcell. In another embodiment, the cell of the present invention is aninvertebrate cell. In another embodiment, the cell of the presentinvention is a vertebrate cell. In another embodiment, the cell of thepresent invention is an insect cell. In another embodiment, the cell ofthe present invention is an amphibian cell. In another embodiment, thecell of the present invention is a reptile cell. In another embodiment,the cell of the present invention is a mammalian cell.

In another embodiment, the present invention provides a tissue sectionin which individual cells are analyzed.

In one embodiment, the present invention provides a method for theselective capture of a SMN complex component with a ligand. In anotherembodiment, the ligand of the invention binds a Gem. In anotherembodiment, the ligand of the invention selectively binds a Gem. Inanother embodiment, the ligand of the invention binds a Gemin protein.In another embodiment, the ligand of the invention selectively binds aGemin protein.

In another embodiment, the ligand of the present invention is anantibody. In another embodiment, the ligand is a polyclonal antibody. Inanother embodiment, the ligand is a monoclonal antibody. In anotherembodiment, the monoclonal antibody is a monovalent Fab fragments. Inanother embodiment, the monoclonal antibody is a Bivalentmini-antibodies (equivalent to F(ab′)2 fragments). In anotherembodiment, the monoclonal antibody is comprised of a genetic fusion toa variety of common protein tags.

In another embodiment, the antibody of the present invention is amultiple engineered specificity antibody. In another embodiment,bispecific antibodies contain two different binding specificities fusedtogether. In another embodiment, bispecific antibodies of the inventionbind to two adjacent epitopes on a single target antigen. In anotherembodiment, bispecific antibodies of the invention bind to two adjacentepitopes on the SMN complex. In another embodiment, bispecificantibodies of the invention cross-link two different antigens. Inanother embodiment, Bispecific antibodies of the present invention areproduced by fusion of two hybridoma cell lines into a single ‘quadroma’cell line. In another embodiment, effective methods to couple twodifferent Fab modules of the invention incorporate either chemicalconjugation. In another embodiment, effective methods to couple twodifferent Fab modules of the invention incorporate either geneticconjugation. In another embodiment, effective methods to couple twodifferent Fab modules of the invention incorporate fusion to adhesiveheterodimeric domains.

In another embodiment, the antibody of the present invention is abifunctional antibodie. In another embodiment, the antibody of thepresent invention is fused to radio labeled moiety. In anotherembodiment, the antibody of the present invention is fused to an enzyme.

In another embodiment, the antibody of the present invention is derivedfrom antibody libraries. In another embodiment, the antibody of thepresent invention is a specific high-affinity antibody selected bylinking phenotype (binding affinity) to genotype, thereby allowingsimultaneous recovery of the gene encoding the selected antibody.

In another embodiment, the antibody of the present invention iscomprised of single-chain Fv fragments. In another embodiment, thesingle-chain Fv fragments antibody of the present invention contain acomplete binding site and consist of the individual heavy and lightchain V domain (12-14 kDa each). In another embodiment, the single-chainFv fragments antibody of the present invention is linked to a singleprotein by a hydrophilic and flexible polypeptide linker. In anotherembodiment, the single-chain Fv fragments antibody of the presentinvention additionally include also a His tag. In another embodiment,the single-chain Fv fragments antibody of the present inventionadditionally includes an immuno-detection epitope. In anotherembodiment, the single-chain Fv fragments antibody of the presentinvention additionally includes a protease specific cleavage site. Inanother embodiment, the linker of the variable region domains (carboxylter-minus of the VL sequence to the amino terminus of the VH sequence)has to be long enough to span the distance from the C-terminus of onedomain to the N-terminus of the second domain (about 3.5 nm).

In another embodiment, the antibody according to the methods of thepresent invention binds a protein within the SMN complex. In anotherembodiment, the antibody according to the methods of the presentinvention is a monoclonal anti-SMN protein antibody. In anotherembodiment, the antibody according to the methods of the presentinvention is the anti-SMN monoclonal antibody 2B1. In anotherembodiment, the 2B1 antibody of the present invention binds Gems. Inanother, embodiment, 2B1 antibody binds cytoplasmic Gems. In another,embodiment, 2B1 antibody binds nuclear Gems (See example 1). In anotherembodiment, the antibody of the present invention binds Gemin1. Inanother embodiment, the antibody of the present invention binds Gemin2.In another embodiment, the antibody of the present invention bindsGemin3. In another embodiment, the antibody of the present inventionbinds Gemin4. In another embodiment, the antibody of the presentinvention binds Gemin5. In another embodiment, the antibody of thepresent invention binds Gemin6. In another embodiment, the antibody ofthe present invention binds Gemin7 (example 5, FIG. 4).

In another embodiment, the ligand of the present invention is labeled.In another embodiment, the antibody of the present invention is labeled.In another embodiment, the labeled antibody of the present invention isa fluorescent labeled antibody. In another embodiment, the label isAlexa Fluor. In another embodiment, the label is green fluorescentprotein. In another embodiment, the label is Oregon green. In anotherembodiment, the label is Emerald. In another embodiment, the label isAzami Green. In another embodiment, the label is ZsGreen1. In anotherembodiment, the label is a blue fluorescent protein. In anotherembodiment, the label is EBFP. In another embodiment, the label isSapphire. In another embodiment, the label is a cyan fluorescentprotein. In another embodiment, the label is cerulean. In anotherembodiment, the label is ECFP. In another embodiment, the label isAmCyan. In another embodiment, the label is Midoriishi-Cyan. In anotherembodiment, the label is a yellow fluorescent protein. In anotherembodiment, the label is ZsYellow1. In another embodiment, the label isPhiYFP. In another embodiment, the label is Citrine. In anotherembodiment, the label is Venus. In another embodiment, the label is anorange fluorescent protein. In another embodiment, the label isKusabira-Orange. In another embodiment, the label is mOrange. In anotherembodiment, the label is a red fluorescent protein. In anotherembodiment, the label is DsRed. In another embodiment, the label isHcRed. In another embodiment, the label is mPlum. In another embodiment,the label is mRaspberry. In another embodiment, the label is mTomato. Inanother embodiment, the label is mStrawberry. In another embodiment, thelabel is green-to-red fluorescent Dendra.

In another embodiment, the present invention provides a SMN complexcomponent fused to a fluorescent probe. In another embodiment, anidentifiable gene product serves as a distinguishable marker for a SMNcomplex component. In another embodiment, the methods of the presentinvention provide a Gemin protein fused to a fluorescent probe of theinvention. In another embodiment, the methods of the present inventionprovide that a SMN complex component is a chimera comprising a SMNcomplex component and a fluorescent protein.

In another embodiment, the label of the present invention is aradioactive label. In another embodiment, a ligand of the presentinvention is radioactively labeled with ³²P. In another embodiment, anantibody of the present invention is radioactively labeled with ³²P. Inanother embodiment, a ligand of the present invention is radioactivelylabeled with ¹²⁵I. In another embodiment, an antibody of the presentinvention is radioactively labeled with ¹²⁵I. In another embodiment, thelabel of the present invention is a chemiluminescent label. In anotherembodiment, the label of the present invention is a gold label.

In some embodiments, the detection method is indirect comprising aligand of the present invention similar to immunohistochemical probes asknown to one skilled in the art. In another embodiment, probes may belabeled with hapten or biotin used to bring an enzyme which creates adetectable event (e.g., chemiluminescent, colorimetric or fluorescent)to the SMN complex component site. In another embodiment, whereinamplification of the detection signal is required, a secondary labeledantibody specifically identifying the primary antibody is utilized. Inanother embodiment, the methods utilizing a specific probe comprising anantibody enable selective identification of an SMN component.

In another embodiment, the method of the present invention providesmeasuring the level of an SMN complex component relocalization from thecytoplasm to the nucleus. In another embodiment, the method of thepresent invention provides measuring an SMN complex component in thenucleus and cytoplasm of said cell. In another embodiment, the method ofthe present invention provides measuring an absolute number of an SMNcomplex component in the nucleus and cytoplasm.

In one embodiment, the invention provides a method of identifying an SMNcomplex component, which comprises visualizing the probed SMN complexcomponent. In another embodiment, visualization of SMN complex componentis carried out by exposing the labeled specimen to a film. In anotherembodiment, visualization of SMN complex component is performed using afluorescent microscope. In another embodiment, visualization of SMNcomplex component is performed using a confocal microscope. In anotherembodiment, visualization of SMN complex component is performed using atransmission electron microscope (TEM). In another embodiment,visualization of SMN complex component is performed using a scanningelectron microscope (SEM). In another embodiment, visualization of SMNcomplex component is performed using a reflection electron microscope(REM). In another embodiment, visualization of SMN complex component isperformed using a scanning transmission electron microscope (STEM). Inanother embodiment, a light microscope is used for visualization of SMNcomplex component, while in another embodiment; the signal is detectableusing the naked eye. In another embodiment, the results of the abovementioned visualization methods can be further recorded and/orvisualized on a charge-coupled device (CCD) camera. In anotherembodiment, a back-illuminated, cooled, CCD camera is used forluminescent detection. In another embodiment, color CCD cameras as wellas dual-photon lasers are used for ultra-high-resolution imaging of afluorescent protein.

In another embodiment, the invention provides means of quantifying theprobed SMN complex component. In another embodiment, quantification isassessed by a fluorometer. In another embodiment digital images arecollected. In another embodiment digital images are collected from atleast two fields in each well. In another embodiment digital images arecollected from at least three fields in each well. In another embodimentdigital images are collected from at least four fields in each well. Inanother embodiment digital images are collected from at least fourfields in each well. In another embodiment digital images are collectedfrom at least five fields in each well. In another embodiment digitalimages are collected from six fields in each well (example 2).

In another embodiment, the methods of the present invention providemeans of analyzing the digital images of the invention. In anotherembodiment, means of analyzing the digital images comprise computersoftware based on algorithms for multiple parameters. In anotherembodiment, the methods of the present invention provide that multipleparameters comprise at least two parameters. In another embodiment,multiple parameters comprise relative nuclear and cytoplasmic florescentintensities (example 2). In another embodiment, multiple parameterscomprise relative nuclear and cytoplasmic chemiluminescent or gold labelintensities. In another embodiment, multiple parameters compriserelative nuclear and cytoplasmic radioactive label intensities. Inanother embodiment, multiple parameters further comprise number, sizeand signal intensity of an SMN complex component. In another embodiment,multiple parameters further comprise number, size and signal intensityof Gems.

In another embodiment, chemiluminescent detection comprises an enzymewhich provides enzymatic amplification. In another embodiment, theenzyme which provides enzymatic amplification is horseradish peroxidase.In another embodiment, the enzyme which provides enzymatic amplificationis alkaline phosphatase. In another embodiment, the enzyme whichprovides enzymatic amplification is β-galactosidase.

In another embodiment, the method of the present invention providesnucleus boundaries identification. In another embodiment, the method ofthe present invention provides that nucleus boundaries are identifiedaccording to nuclear membrane staining. In another embodiment, themethod of the present invention provides a separate channel to definethe boundary of each nucleus. In another embodiment, the method of thepresent invention provides that DAPI-stained images are collected in aseparate channel to define the boundary of each nucleus (example 1). Inanother embodiment a change in SMN sub-cellular localization is examineddirectly (FIG. 2).

In another embodiment, the present invention provides a method ofquantitatively measuring the amount of a SMN complex component in thenucleus and in the cytoplasm. In another embodiment, the presentinvention provides a method for comparative assessment of the relativeamounts of a SMN complex component in the nucleus and in the cytoplasm.

In another embodiment, the present invention provides a method forscreening a p38 MAPK modifier effective in a particular cell type. Inanother embodiment, the present invention provides a method forscreening a p38 MAPK modifier effective in a particular disease.

In another embodiment, a p38 MAPK inducer is a p38 MAPK inducer. Inanother embodiment, a p38 MAPK inducer of the present invention acts asa tumor suppressor. In another embodiment, a p38 MAPK inducer acts as acell growth inhibitor of human breast cancer cells and thus treatsbreast cancer. In another embodiment, a p38 MAPK inhibitor of thepresent invention treats Alzheimer's disease. In another embodiment, ap38 MAPK inhibitor of the present invention treats early stages ofAlzheimer's disease. In another embodiment, a p38 MAPK inhibitor of thepresent invention treats amyotrophic lateral sclerosis (ALS or LouGehrig's disease). In another embodiment, a p38 MAPK inhibitor of thepresent invention treats early stages of ALS (Lou Gehrig's disease).

In another embodiment, a p38 MAPK inhibitor of the present inventionsuppresses replication of Encephalomyocarditis virus. In anotherembodiment, a p38 MAPK inhibitor of the present invention suppressesHepatitis C virus signaling. In another embodiment, a p38 MAPK inhibitorof the present invention can abolish cytotoxic T-cell (CTL) bystanderkilling by HIV-1-infected macrophages and thus may treat subjectsinfected with HIV.

In another embodiment, the kit of present invention comprises a ligandselective for a SMN complex component. In another embodiment, the kit ofpresent invention comprises SMN complex component relocalizationmeasuring reagents. In another embodiment, the kit of present inventioncomprises a ligand selectively capturing a SMN complex component. Inanother embodiment, the ligand of the invention binds Gem. In anotherembodiment, the ligand of the invention specifically binds a Geminprotein.

In another embodiment, the kit of the present invention comprises anantibody. In another embodiment the kit of the present inventioncomprises a polyclonal antibody. In another embodiment, the kit of thepresent invention comprises a monoclonal antibody. In anotherembodiment, the kit of the present invention comprises a monovalent Fabfragments. In another embodiment, the kit of the present inventioncomprises a Bivalent mini-antibody.

In another embodiment, the kit of the present invention comprises amultiple engineered specificity antibody. In another embodiment, the kitof the present invention comprises a bispecific antibodies which containtwo different binding specificities fused together. In anotherembodiment, bispecific antibodies of the invention bind to two adjacentepitopes on a single target antigen. In another embodiment, bispecificantibodies of the invention bind to two adjacent epitopes on the SMNcomplex. In another embodiment, bispecific antibodies of the inventioncross-link two different antigens. In another embodiment, bispecificantibodies of the present invention are produced by fusion of twohybridoma cell lines into a single ‘quadroma’ cell line. In anotherembodiment, effective methods to couple two different Fab modules of theinvention incorporate either chemical conjugation. In anotherembodiment, effective methods to couple two different Fab modules of theinvention incorporate either genetic conjugation. In another embodiment,effective methods to couple two different Fab modules of the inventionincorporate fusion to adhesive heterodimeric domains.

In another embodiment, the kit of the present invention comprises abifunctional antibody. In another embodiment, the antibody of thepresent invention is fused to radio labeled moiety. In anotherembodiment, the antibody of the present invention is fused to an enzyme.

In another embodiment, the kit of the present invention comprises anantibody derived from antibody libraries. In another embodiment, the kitof the present invention comprises an antibody which is a specifichigh-affinity antibody selected by linking phenotype (binding affinity)to genotype, thereby allowing simultaneous recovery of the gene encodingthe selected antibody.

In another embodiment, the kit of the present invention comprises anantibody composed of a single-chain Fv fragments. In another embodiment,the single-chain Fv fragments antibody of the present invention containa complete binding site and consist of the individual heavy and lightchain V domain. In another embodiment, the single-chain Fv fragmentsantibody of the present invention is linked to a single protein by ahydrophilic and flexible polypeptide linker. In another embodiment, thesingle-chain Fv fragments antibody of the present invention additionallyinclude also a His tag. In another embodiment, the single-chain Fvfragments antibody of the present invention additionally includes animmunodetection epitope. In another embodiment, the single-chain Fvfragments antibody of the present invention additionally includes aprotease specific cleavage site. In another embodiment, the linker ofthe variable region domains (carboxyl ter-minus of the VL sequence tothe amino terminus of the VH sequence) has to be long enough to span thedistance from the C-terminus of one domain to the N-terminus of thesecond domain (about 3.5 nm).

In another embodiment, the kit of the present invention comprises anantibody which binds a protein within the SMN complex. In anotherembodiment, the kit of the present invention comprises a monoclonalanti-SMN component antibody. In another embodiment, the kit of thepresent invention comprises an anti-SMN monoclonal 2B1 antibody. Inanother embodiment, the kit of the present invention comprises 2B1antibody which binds Gems. In another embodiment, the kit of the presentinvention comprises 2B1 antibody which binds cytoplasmic Gems. Inanother, embodiment, the kit of the present invention comprises 2B1antibody which binds nuclear Gems. In another embodiment, the kit of thepresent invention comprises an anti-Gemin1 antibody. In anotherembodiment, the kit of the present invention comprises an anti-Gemin1antibody. In another embodiment, the kit of the present inventioncomprises an anti-Gemin2 antibody. In another embodiment, the kit of thepresent invention comprises an anti-Gemin3 antibody. In anotherembodiment, the kit of the present invention comprises an anti-Gemin4antibody. In another embodiment, the kit of the present inventioncomprises an anti-Gemin5 antibody. In another embodiment, the kit of thepresent invention comprises an anti-Gemin6 antibody. In anotherembodiment, the kit of the present invention comprises an anti-Gemin7antibody.

In another embodiment, the kit of the present invention comprises alabeled ligand. In another embodiment, the kit of the present inventioncomprises a labeled antibody. In another embodiment, the labeledantibody is a fluorescent labeled antibody as described hereinabove.

In another embodiment, the kit of the present invention comprises aplasmid encoding a SMN complex component fused to a fluorescent probe.In another embodiment, the fluorescent probe is selected from thefluorescent proteins described hereinabove. In another embodiment,plasmid comprises a Gemin protein fused to a fluorescent probe of theinvention.

In another embodiment, the kit of the present invention comprises aradioactive label. In another embodiment, the kit of the presentinvention comprises a ligand radioactively labeled with ³²P. In anotherembodiment, the kit of the present invention comprises an antibodyradioactively labeled with ³²P. In another embodiment, the kit of thepresent invention comprises a ligand radioactively labeled with ¹²⁵I. Inanother embodiment, the kit of the present invention comprises anantibody radioactively labeled with ¹²⁵I. In another embodiment, the kitof the present invention comprises a ligand attached to chemiluminescentlabel. In another embodiment, the kit of the present invention comprisesan antibody attached to a chemiluminescent label. In another embodiment,the kit of the present invention comprises a ligand conjugated to achemiluminescent label. In another embodiment, the kit of the presentinvention comprises an antibody conjugated to a chemiluminescent label.In another embodiment, the kit of the present invention comprises aligand conjugated to a gold label. In another embodiment, the kit of thepresent invention comprises an antibody conjugated to a gold label.

In another embodiment, the kit of the present invention comprisesbuffers. In another embodiment, buffers of the present inventioncomprise the reagents of the present invention. In another embodiment,the kit of the present invention comprises cell fixation reagents. Inanother embodiment, the fixation reagent of the present invention is analcohol. In another embodiment, the fixation reagent of the presentinvention is methanol. In another embodiment, the fixation reagent ofthe present invention is ethanol. In another embodiment, the fixationreagent of the present invention is paraformaldehyde. In anotherembodiment, the fixation reagent of the present invention isglutaraldehyde. In another embodiment, the fixation reagent of thepresent invention is an aldehyde. In another embodiment, the fixationreagent of the present invention is dimethylsuberimidate.

In another embodiment, the kit of the present invention comprises apermeability enhancing agents such as detergents. In another embodiment,the permeability enhancing agent of the present invention is saponin. Inanother embodiment, the permeability enhancing agent of the presentinvention is tween.

In another embodiment, the kit of the present invention comprises anenzyme conjugated to avidin. a ligand labeled with a hapten. In anotherembodiment, the kit of the present invention comprises a biotinylatedligand. In another embodiment, the kit of the present inventioncomprises a label conjugated to avidin. In another embodiment, the kitof the present invention comprises an enzyme conjugated to avidin. Inanother embodiment, the kit of the present invention comprises an enzymewhich creates a detectable event (e.g., chemiluminescent, colorimetricor fluorescent).

In another embodiment, the kit of the present invention comprisesalkaline phosphatase. In another embodiment, the kit of the presentinvention comprises β-galactosidase. In another embodiment, the kit ofthe present invention comprises peroxidase. In another embodiment, thekit of the present invention comprises horseradish peroxidase.

In another embodiment, the kit of the present invention comprises asecondary labeled antibody specifically identifying the primaryantibody.

In another embodiment, the kit of the present invention furthercomprises a nuclear dye. In another embodiment, the kit of the presentinvention further comprises Azur A. In another embodiment, the kit ofthe present invention further comprises hematoxylin. In anotherembodiment, the kit of the present invention further comprises MethylViolet. In another embodiment, the kit of the present invention furthercomprises Phloxine B. In another embodiment, the kit of the presentinvention further comprises Pyronin Y. In another embodiment, the kit ofthe present invention further comprises Safranin O. In anotherembodiment, the kit of the present invention further comprises acytoplasmic dye. In another embodiment, the kit of the present inventionfurther comprises a cytoplasmic membrane dye. In another embodiment, thekit of the present invention further comprises a nuclear membrane dye.In another embodiment, the kit of the present invention furthercomprises DAPI (example 1). In another embodiment, the kit of thepresent invention further comprises a sub-cellular dye as will bereadily known to one of skill in the art.

In another embodiment, the kit of the present invention furthercomprises a multi-well plate. In another embodiment, a multi-well plateof the present invention comprises 96 wells. In another embodiment, amulti-well plate of the present invention comprises 384 wells. Inanother embodiment, a multi-well plate of the present inventioncomprises 1536 wells. In another embodiment, a multi-well plate of thepresent invention comprises from 2-5000 wells. In another embodiment, amulti-well plate of the present invention comprises from 20-3000 wells.In another embodiment, a multi-well plate of the present inventioncomprises from 96-2000 wells.

In another embodiment, the kit of the present invention comprisesscreening compounds. In another embodiment, the kit of the presentinvention comprises a protein synthesis inhibitor. In anotherembodiment, the kit of the present invention comprises a p38 inducer. Inanother embodiment, the protein synthesis inhibitor is cycloheximide. Inanother embodiment, the protein synthesis inhibitor is Anisomycin. Inanother embodiment, the p38 MAPK inducer is TNFα. In another embodiment,the p38 MAPK inducer is LPS. In another embodiment, the kit of thepresent invention comprises a protein synthesis inhibitor and a p38 MAPKinducer. In another embodiment, the kit of the present inventioncomprises Anisomycin. In another embodiment, the kit of the presentinvention comprises Anisomycin and cycloheximide. In another embodiment,the kit of the present invention comprises Anisomycin and TNFα. Inanother embodiment, the kit of the present invention comprisesAnisomycin and LPS. In another embodiment, the kit of the presentinvention comprises cycloheximide and TNFα. In another embodiment, thekit of the present invention comprises cycloheximide and LPS.

In another embodiment, the system of the present invention screens p38MAPK modifiers. In another embodiment, the system of the presentinvention identifies p38 MAPK inducers. In another embodiment, thesystem of the present invention identifies p38 MAPK inhibitors. Inanother embodiment, the system of the present invention comprises thekit of the present invention as described hereinabove. In anotherembodiment, the system of the present invention comprises visualizationmeans.

In another embodiment, the terms “screen” and “identify” are usedinterchangeably.

In another embodiment, the terms “activator” and “inducer” are usedinterchangeably.

In one embodiment, the system of the present invention comprises anapparatus for exposing the screen of the present invention to a film. Inanother embodiment, the system of the present invention comprises afluorescent microscope. In another embodiment, the system of the presentinvention comprises a confocal microscope. In another embodiment, thesystem of the present invention comprises a transmission electronmicroscope. In another embodiment, the system of the present inventioncomprises a scanning electron microscope. In another embodiment, thesystem of the present invention comprises a reflection electronmicroscope. In another embodiment, the system of the present inventioncomprises a scanning transmission electron microscope. In anotherembodiment, the system of the present invention comprises a lightmicroscope. In another embodiment, the system of the present inventioncomprises a charge-coupled device (CCD) camera. In another embodiment,the system of the present invention comprises a back-illuminated,cooled, CCD camera. In another embodiment, the system of the presentinvention comprises a color CCD camera. In another embodiment, thesystem of the present invention comprises a dual-photon laser.

In another embodiment, the system of the present invention comprises afluorometer. In another embodiment, the system of the present inventioncomprises a computer which digitally records the microscopicallycaptured images. In another embodiment, the system of the presentinvention comprises a computer which digitally records the CCD cameracaptured images.

In another embodiment, the system of the present invention comprisescomputer software. In another embodiment, the computer software analyzesthe digital images collected from at least two fields in each well. Inanother embodiment, the computer software analyzes the digital imagescollected from at least three fields in each well. In anotherembodiment, the computer software analyzes the digital images collectedfrom at least four fields in each well. In another embodiment, thecomputer software analyzes the digital images collected from at leastfive fields in each well. In another embodiment, the computer softwareanalyzes the digital images collected from at least six fields in eachwell. In another embodiment, the computer software analyzes the digitalimages collected from six fields in each well.

In another embodiment, the system of the present invention comprisescomputer software based on algorithms for multiple parameters. Inanother embodiment, multiple parameters comprise at least twoparameters. In another embodiment, multiple parameters comprise relativenuclear and cytoplasmic florescent intensities. In another embodiment,multiple parameters comprise relative nuclear and cytoplasmicchemilurminescent or gold label intensities. In another embodiment,multiple parameters comprise relative nuclear and cytoplasmicradioactive label intensities. In another embodiment, multipleparameters further comprise number, size and signal intensity of a SMAcomplex component. In another embodiment, multiple parameters furthercomprise number, size and signal intensity of Gems.

In another embodiment, solid carriers/diluents for use in methods andcompositions of the present invention include, but are not limited to, agum, a starch (e.g. corn starch, pregeletanized starch), a sugar (e.g.,lactose, mannitol, sucrose, dextrose), a cellulosic material (e.g.microcrystalline cellulose), an acrylate (e.g. polymethylacrylate),calcium carbonate, magnesium oxide, talc, or mixtures thereof.

In another embodiment, the compositions further comprise binders (e.g.acacia, cornstarch, gelatin, carbomer, ethyl cellulose, guar gum,hydroxypropyl cellulose, hydroxypropyl methyl cellulose, povidone),disintegrating agents (e.g. cornstarch, potato starch, alginic acid,silicon dioxide, croscarmelose sodium, crospovidone, guar gum, sodiumstarch glycolate), buffers (e.g., Tris-HCI, acetate, phosphate) ofvarious pH and ionic strength, additives such as albumin or gelatin toprevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80,Pluronic F68, bile acid salts), protease inhibitors, surfactants (e.g.sodium lauryl sulfate), permeation enhancers, solubilizing agents (e.g.,glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite, butylated hydroxyanisole), stabilizers (e.g.hydroxypropyl cellulose, hyroxypropylmethyl cellulose), viscosityincreasing agents (e.g. carbomer, colloidal silicon dioxide, ethylcellulose, guar gum), sweeteners (e.g. aspartame, citric acid),preservatives (e.g., Thimerosal, benzyl alcohol, parabens), lubricants(e.g. stearic acid, magnesium stearate, polyethylene glycol, sodiumlauryl sulfate), flow-aids (e.g. colloidal silicon dioxide),plasticizers (e.g. diethyl phthalate, triethyl citrate), emulsifiers(e.g. carbomer, hydroxypropyl cellulose, sodium lauryl sulfate), polymercoatings (e.g., poloxamers or poloxamines), coating and film formingagents (e.g. ethyl cellulose, acrylates, polymethacrylates) and/oradjuvants. Each of the above excipients represents a separate embodimentof the present invention.

In some embodiments, the dosage forms of the present invention areformulated to achieve an immediate release profile, an extended releaseprofile, or a delayed release profile. In some embodiments, the releaseprofile of the composition is determined by using specific excipientsthat serve for example as binders, disintegrants, fillers, or coatingmaterials. In one embodiment, the composition will be formulated toachieve a particular release profile as known to one skilled in the art.

In one embodiment, the composition is formulated as an oral dosage form.In one embodiment, the composition is a solid oral dosage formcomprising tablets, chewable tablets, or capsules. In one embodiment thecapsules are soft gelatin capsules. In another embodiment, capsules asdescribed herein are hard-shelled capsules. In another embodiment,capsules as described herein are soft-shelled capsules. In anotherembodiment, capsules as described herein are made from gelatine. Inanother embodiment, capsules as described herein are made fromplant-based gelling substances like carrageenans and modified forms ofstarch and cellulose.

In other embodiments, controlled- or sustained-release coatings utilizedin methods and compositions of the present invention include formulationin lipophilic depots (e.g. fatty acids, waxes, oils).

The compositions also include, in another embodiment, incorporation ofthe active material into or onto particulate preparations of polymericcompounds such as polylactic acid, polglycolic acid, hydrogels, etc, oronto liposomes, microemulsions, micelles, unilamellar or multilamellarvesicles, erythrocyte ghosts, or spheroplasts.) Such compositions willinfluence the physical state, solubility, stability, rate of in vivorelease, and rate of in vivo clearance. In another embodiment,particulate compositions of the active ingredients are coated withpolymers (e.g. poloxamers or poloxamines)

In another embodiment, the compositions containing the compounds of thepresent invention are delivered in a vesicle, e.g. a liposome (seeLanger, Science 249:1527-1533 (1990); Treat et al., in Liposomes in theTherapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler(eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.317-327; see generally ibid). In another embodiment, the compositionscontaining the Exenatide and omega-3 fatty acid are delivered in avesicle, e.g. a liposome (see Langer, Science 249:1527-1533 (1990);Treat et al., in Liposomes in the Therapy of Infectious Disease andCancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365(1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid).

The preparation of pharmaceutical compositions that contain an activecomponent, for example by mixing, granulating, or tablet-formingprocesses, is well understood in the art. The active therapeuticingredient is often mixed with excipients that are pharmaceuticallyacceptable and compatible with the active ingredient. For oraladministration, the active ingredients of compositions of the presentinvention are mixed with additives customary for this purpose, such asvehicles, stabilizers, or inert diluents, and converted by customarymethods into suitable forms for administration, such as tablets, coatedtablets, hard or soft gelatin capsules, aqueous, alcoholic or oilysolutions.

Each of the above additives, excipients, formulations and methods ofadministration represents a separate embodiment of the presentinvention.

In one embodiment, the term “treating” refers to curing a disease. Inanother embodiment, “treating” refers to preventing a disease. Inanother embodiment, “treating” refers to reducing the incidence of adisease. In another embodiment, “treating” refers to amelioratingsymptoms of a disease. In another embodiment, “treating” refers toinducing remission. In another embodiment, “treating” refers to slowingthe progression of a disease.

EXPERIMENTAL DETAILS SECTION Materials and Experimental Methods

Compounds

A library of about 5,000 pure bioactive chemicals that includesFDA-approved drugs, known inhibitors and activators of diverse enzymesand receptors, and pure natural compounds was assembled from commercialsources (Microsource Diversity, Tocris, Sigma/Aldrich and othersuppliers). Cycloheximide, trichostatin A, valproic acid and scriptaidwere purchased from Sigma Chemical Co. HDAC inhibitor I and apicidinwere from EMD biosciences.

Cell Culture and Treatments

HeLa PV were cultured in Dulbecco's modified Eagle's medium (Invitrogen)supplemented with 10% fetal bovine serum (Invitrogen). For screening,cells were seeded onto 384-well plates or, for specific experiments, onglass coverslips the day before treatment. Compounds were added directlyinto the culture medium to the indicated concentration and the cellswere then maintained at 37° C. in a 5% CO2 humidified atmosphere for theduration of the treatment. Dispensing of compounds and other automaticliquid handling steps were performed with a Beckman FX multi-channelsystem equipped with Hudson Crane robotic systems.

Antibodies

The following mouse monoclonal antibodies were used for indirectimmunofluorescence: 2B1 (anti-SMN), 2E17 (anti-Gemin2), 12H12(anti-Gemin3), 10G1 (anti-GeminS), 6E2 (anti-Gemin7), 10E10 (anti-PABP),6BG10 (anti-FXR1), Y12 (anti-Sm) and 9H10 (anti-hnRNP A1). Anaffinity-purified rabbit polyclonal antibody was used to detect Gemin6.

Indirect Immunofluorescence Microscopy

Indirect immunofluorescence on HeLa PV cells was performed as follows:HeLa cells, plated on glass coverslips, were briefly washed with PBS,fixed in 2% formaldehyde/PBS for 20 minutes at room temperature,permeabilized in 0.5% Triton X-100/PBS for 5 min at room temperature.Cells were blocked in 3% bovine serum albumin for 1 hour at roomtemperature. Double-label immunofluorescence experiments were performedby separate sequential incubations of each primary antibody, diluted inPBS containing 3% bovine serum albumin, followed by the specificsecondary coupled to fluorescein isothiocyanate or TXRD. All incubationswere at room temperature for 1 hour. Indirect epifluorescence microscopywas performed with a Nikon Eclipse E800 microscope. Digital images werecollected with a Cook Sensicam high performance camera and processedwith the IP laboratory software. Processing of samples forimmunofluorescence microscopy on cells cultured in 384-well plates wasautomated and performed with the aid of microplate washers (ELX405,Bio-Tek) in a similar manner to that previously used for individualsamples on glass slides. The cells were also stained with DAPI to allowdefinition of the nucleus in each cell. Digital images were acquiredwith an automated microscopy and analysis system (IN Cell Analyzer 1000,GE Healthcare). Images were analyzed using automated algorithms withparameters set to calculate mean pixel intensity in each nucleus(defined by DAPI staining and acquired in a separate channel),cytoplasm, and total cell, as well as the relevant calculated ratios ofthese values.

RNA Interference

Transient RNAi was performed as follows: 21-nt RNA duplexes (siRNAs)were designed to target SMN or Gemin2-6 mRNAs. For each targettranscript, at least five siRNAs were designed and tested and, ingeneral, found to behave similarly in all subsequent analyses. Thefollowing siRNA sequences yielded the most efficient protein knockdownsof each target: GAAGAAUACUGCAGCUUCC (SEQ ID No. 8) for SMN;GCAGCUCAAUGUCCAGAUG (SEQ ID No. 9) for Gemin2; GGCUUAGAGUGUCAUGUCU (SEQID No. 10) for Gemin3; ACUCCCCAGUGAGAC CAUU (SEQ ID No. 11) for Gemin4;GCAUAGUGGUGAUAAUUGA (SEQ ID No. 12) for Gemin5 and AACUACAGACCCAGUCUCUGC(SEQ ID No. 13) for Gemin6. In addition, a siRNA initially designed totarget Y14, a component of the exon junction complex, which failed toproduce any protein reduction within 44 h, was used as a control(CCCGGACCACAACGCUCUG, SEQ ID No. 14). All siRNAs were chemicallysynthesized and purified by Dharmacon Research. Transfections of siRNAsinto HeLa PV cells were performed using Oligofectamine™ (Invitrogen) asspecified by the manufacturer. Transfected cells were analyzed 40-44 hpost-transfection.

Example 1: SMN Complex Composition

In order to determine whether Gems are affected by changes in thecomposition of the complex, the amount of several Gemins was reduced,one at a time, by RNA interference. The morphology of Gems was monitoredby immunofluorescence microscopy using the anti-SMN monoclonal antibody2B1.

Reduction of Gemin3 or Gemin4 caused large changes in the size, numberand SMN signal intensity of Gems (FIG. 1a ). The effect of thetreatments was quantified with software analysis of the collected imagesfrom a large number of cells. The results indicated a two-fold increasein both signal intensity as well as gem number in each case (FIG. 1b ).Thus, changes in the SMN complex are reflected by changes in themorphology of Gems.

Example 2: Screening Assay

Based on the results obtained in Example 1, a library of ˜5,000biologically active small molecules was screened on HeLa cells in384-well plates. Each well was incubated with a different compound (at10 M) and the cells were processed for indirect immunofluorescence.Digital images were collected from six fields in each well and analyzedusing imaging algorithms for multiple parameters including relativenuclear and cytoplasmic intensities, and number, size and signalintensity of Gems. DAPI-stained images were collected in a separatechannel to define the boundary of each nucleus. Images of individualfields in wells were flagged by the software as showing a significantchange in SMN sub-cellular localization. These individual wells wereexamined directly, and active compounds were re-tested for verification(FIG. 2).

Example 3: Redistribution of SMN from the Cytoplasm to the Nucleus

The previous Examples screened compounds that caused changes in Gems.However, it was noticed that several compounds produced a massiveredistribution of SMN from the cytoplasm to the nucleus within arelatively short time (2-6 hours) of treatment (according to thematerials and methods of example 2). The most striking accumulation ofSMN in the nucleus was observed after treatment with the commonly usedprotein synthesis inhibitor, cycloheximide (CHX). The effect of CHX wasevident after only 2 hours of treatment and there was little SMNdetected in the cytoplasm after 8 or more hours of treatment. CHX effecton redistribution of SMN from the cytoplasm to the nucleus wasconcentration-dependent exhibiting a clear effect even at low micromolarconcentrations (0.1-40 μM) (FIG. 3a, b ). The nuclear accumulation ofSMN did not require complete inhibition of protein synthesis and wasproportional to the extent of inhibition of protein synthesis. Aninactive cycloheximide analog, cycloheximide-N-ethylethanoate, did notshow any effect (FIG. 8).

The accumulation of SMN in the nucleus and its disappearance from thecytoplasm under conditions of protein synthesis inhibition indicatesthat it represents SMN protein that translocated from the cytoplasm.Other protein synthesis inhibitors, including emetine, an irreversibleinhibitor of translation elongation, and the peptide chain terminator,puromycin, showed the same effect as cycloheximide, demonstrating thatit is a general consequence of protein synthesis inhibitors.

The effect of several compounds that do not directly inhibittranslation, but rather cause translation inhibition as an indirecteffect was monitored. Inducers of endoplasmic reticulum stress, such asthapsigargin and tunicamycin elicit the unfolded protein response, whichleads to a reduction in general protein translation through thephosphorylation of eIF2α17. Treatment of cells with thapsigargin ortunicamycin caused SMN to accumulate in the nucleus (FIG. 8), althoughthe effect was not as complete as that seen after treatment with highlevels of CHX, consistent with the fact that protein synthesis is notshut down and selective translation of a class of mRNAs continues underthese conditions.

Example 4: The Protein Synthesis Inhibitor-Induced Nuclear Accumulationof SMN is Specific

The protein synthesis inhibitor-induced nuclear accumulation of SMN isnot the result of general cell toxicity as no reduction in cellviability was monitored even after an overnight treatment of these cellswith 10 μM CHX, conditions in which all of the SMN was localized to thenucleus. In addition, the nuclear accumulation is specific to thelocalization of SMN and not the result of general mis-localization ofproteins, as the localization of many other, both nuclear andcytoplasmic proteins that were examined, including the RNA-bindingproteins hnRNP A1, poly(A)-binding protein (PABP), FXR1 and snRNPs, wasnot significantly affected (FIG. 3c ).

Furthermore, the effect was reversible as SMN staining graduallyre-appeared in the cytoplasm when cells were washed and placed in freshmedium devoid of cycloheximide. A similar effect of protein synthesisinhibitors was also observed in several other cell types and in otherspecies, including U2OS cells and both human and mouse fibroblasts, andwas independent of the amount of SMN they contained. Hela cells withreduced SMN by RNAi, compared to control cells expressing anon-targeting shRNA, showed a similar effect (FIG. 9).

Example 5: Gemins Also Relocate to the Nucleus

According to the materials and methods of Example 2, treated cells werestained with antibodies to each of the Gemins. The results indicate thatin addition to SMN, several of the Gemins, with the exception of Gemin3and Gemin5, also relocated to the nucleus (FIG. 4).

Thus, CHX treatment causes most of the components of the SMN complex toaccumulate in the nucleus, and not just SMN alone. This furtherindicates that the response is a specific effect upon SMN and someassociated proteins. Furthermore, the total amount of SMN in HeLa cellstreated with CHX was similar to that found in untreated cells, showingthat the SMN complexes that accumulated in the nucleus must have beentranslocated from the cytoplasm.

These observations were made after relatively short treatment times (4-6hours). However, at longer times (>24 hr), nuclear translocation of SMNwas observed for several other compounds. Notably, histone deacetylase(HDAC) inhibitors, including valproic acid (VPA), trichostatin A,scriptaid, apicidin and HDAC inhibitor 1, that caused the same effect asprotein synthesis inhibitors (FIG. 5a ).

These compounds showed strong toxicity and resulted in a large reductionin cell numbers. However, they do not cause inhibition of proteinsynthesis, indicating that their effect on the translocation of SMNworks by a different, although possibly convergent, mechanism from thatof the protein synthesis inhibitors.

VPA also displayed a clear concentration dependence effect on thenuclear localization of SMN in addition to its specificity inrelocalizing a subset of the SMN complex to the nucleus (FIGS. 5b and c). Namely, Gemin3 and Gemin5 remain predominantly cytoplasmic upon a 24hour treatment with VPA. This further shows that SMN likely responds tothese compounds in a similar manner.

Thus these experiments demonstrate that the cellular localization of theSMN complex is a dynamic and highly regulated process. Specifically,reduction in protein synthesis as a result of either direct interactionof inhibitors with the translation machinery or indirectly, as aconsequence of ER stress, profoundly affects the SMN complex,translocating SMN and several of the complex components to the nucleus.

Although both translation inhibitors and HDAC inhibitors show a similareffect, the former class of compounds elicited the most rapid responses.The noticeable increase in the amount of an SMN complex component in thenucleus, even at low doses of inhibitor that produce only a very smallreduction in overall translation, indicates that even mild stresses thatattenuate translation have an effect on the balance of the SMN complex.

Example 6: p38 MAPK Activators Antagonise Protein Synthesis InhibitorsInduced SMN Relocalization from the Cytoplasm to the Nucleus

The previous examples screened compounds that produced a massiveredistribution of SMN from the cytoplasm to the nucleus within arelatively short time (2-6 hours) of treatment (according to thematerials and methods of example 2). The most striking accumulation ofSMN in the nucleus was observed after treatment with the commonly usedprotein synthesis inhibitor, cycloheximide (CHX).

The accumulation of SMN in the nucleus and its disappearance from thecytoplasm under conditions of protein synthesis inhibition indicatesthat it represents SMN protein that translocated from the cytoplasm.

Next, the effect of p38 MAPK activators and inhibitors on SMN complexcomponents relocalization under conditions of protein synthesisinhibition was monitored. Administration of 10 μM SB202190 incombination with either 10 μM CHX or 1 μg/ml Anisomycin changeconcentrations as above resulted in accumulation of SMN complexcomponents in the nucleus of the treated HeLa cells (FIG. 6).Administration of 150 ng/ml TNFα resulted in accumulation of SMN complexcomponents in the cytoplasm of the treated HeLa cells (FIG. 6). When 150ng/ml TNFα was combined with 10 μM CHX SMN complex component stillremained in the cytoplasm of the treated HeLa cells (FIG. 6).

Thus, a p38 MAPK inducer such as TNFα antagonizes the impact of theprotein inhibitor CHX on translocation of SMN complex component from thecytoplasm to the nucleus. When p38 MAPK inhibitor-SB202190 wasco-administered with CHX, SMN complex relocalized from the cytoplasm tothe nucleus. Thus, in the absence of p38 MAPK activation, whichantagonizes the effect of protein synthesis inhibitors, the SMN complexrelocalizes from the cytoplasm to the nucleus.

What is claimed is:
 1. A method for testing a candidate compound for anability to decrease p38 mitogen-activated protein kinase (MAPK)enzymatic activity in a cell, comprising the steps of: contacting saidcell with said candidate compound and an additional compound, whereinsaid additional compound is capable of inhibiting protein synthesis insaid cell and increasing an enzymatic activity of said p38 MAPK in saidcell; contacting a control cell with said additional compound in theabsence of said candidate compound; and in both the cytoplasm andnucleus of said cell and said control cell, whereby, if, relative to thecontrol cell, said candidate compound increases the amount of the SMNcomplex component in said nucleus, decreases the amount of the SMNcomplex component in said cytoplasm, or a combination thereof, then saidcompound exhibits an ability to decrease MAPK enzymatic activity.
 2. Themethod of claim 1, wherein said compound capable of inhibiting proteinsynthesis in said cell and increasing an enzymatic activity of said MAPKin said cell is anisomycin.
 3. The method of claim 1, wherein said cellis a eukaryotic cell.
 4. The method of claim 1, wherein said methodfurther comprises the step of contacting said cell with a ligandselective for an SMN complex component.
 5. The method of claim 4,wherein said ligand is an antibody.
 6. The method of claim 5, whereinsaid antibody is a monoclonal antibody, a labeled antibody or both. 7.The method of claim 4, wherein said SMN complex component is fused to afluorescent probe.
 8. The method of claim 4, wherein said SMN complexcomponent is a Gem, or a Gemin.
 9. The method of claim 8, wherein saidGemin is Gemin2, Genin6, Gemin7, or any combination thereof.
 10. Themethod of claim 1, wherein the step of measuring comprises the use of avisualization means to identify said SMN complex component.
 11. Themethod of claim 10, wherein said visualization means comprise amicroscope.
 12. The method of claim 11, wherein said step ofvisualization of said SMN complex component further comprises the use ofa digital image.
 13. The method of claim 12, wherein said digital imageis analyzed using imaging algorithms for parameters comprising relativenuclear signal intensity and relative cytoplasmic signal intensity.